Humanized anti-CD11a antibodies

ABSTRACT

Humanized anti-CD11a antibodies and various uses therefor are disclosed. The humanized anti-CD11a antibody may bind specifically to human CD11a I-domain, have an IC50(nM) value of no more than about 1 nM for preventing adhesion of Jurkat cells to normal human epidermal keratinocytes expressing ICAM-1, and/or an IC50 (nM) value of no more than about 1 nM in the mixed lymphocyte response assay.

This is a non-provisional application filed under 37 CFR 1.53(b)(1),claiming priority under USC Section 119(e) to provisional ApplicationSerial No. 60/031,971, filed on Nov. 27, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to humanized anti-CD11a antibodies.

2. Description of Related Art

Lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is involvedin leukocyte adhesion during cellular interactions essential forimmunologic responses and inflammation (Larson et al., Immunol. Rev.114:181-217 (1990)). LFA-1 is a member of the β2 integrin family andconsists of a unique α subunit, CD11a, and a β subunit, CD18, common toother β2 integrin receptors Mac-1 and p150,95. The ligands of LFA-1include intercellular adhesion molecule-1, |CAM-1, expressed onleukocytes, endothelium, and dermal fibroblasts (Dustin et al., J.Immunol. 137: 245-254 (1986)), ICAM-2 expressed on resting endotheliumand lymphocytes (de Fougerolles et a., J. Exp. Med. 174: 253-267(1991)), and ICAM-3 expressed on monocytes and resting lymphocytes (deFougerolles et al., J. Exp. Med. 179: 619-629 (1994)).

Monoclonal antibodies (MAbs) against LFA-1 and the ICAMs have beenshown, in vitro, to inhibit several T cell-dependent immune functionsincluding T cell activation (Kuypers et al., Res. Immunol. 140:461(1989)), T cell-dependent B cell proliferation (Fischer et al., J.Immunol. 136: 3198-3203 (1986)), target cell lysis (Krensky et al., J.Immunol. 131: 611-616 (1983)), and adhesion of T cells to vascularendothelium (Lo et al., J. Immunol. 143: 3325-3329 (1989)). In mice,anti-CD11a MAbs induce tolerance to protein antigens (Tanaka et al.,Eur. J. Immunol. 25: 1555-1558 (1995)) and prolong survival of cardiac(Cavazzana-Calvo et al., Transplantation 59: 1576-1582 (1995); Nakakuraet al., Transplantation 55: 412-417 (1993)), bone marrow(Cavazzana-Calvo et al., Transplantation 59: 1576-1582 (1995); vanDijken et al., Transplantation 49: 882-886 (1990)), corneal (He et al.,Invest. Opthamol. Vis. Sci. 35: 3218-3225 (1994)), islet (Nishihara etal., Transplantation Proc. 27: 372 (1995)) and thyroid (Talento et al.,Transplantation 55: 418-422 (1993)) allografts.

In humans, anti-CD11a MAbs prevent graft failure after bone marrowtransplantation (Fischer et al., Blood 77: 249-256 (1991); Stoppa etal., Transplant Intl. 4: 3-7 (1991)) and preliminary clinical studies ofrenal allografts treated prophylactically with anti-CD11a MAb, inaddition to corticosteroids and azathioprine, are promising (Hourmant etal., Transplantation 58: 377-380 (1994)). Current therapies againstgraft rejection include use of OKT3, a murine anti-human CD3 MAb, andcyclosporin A. OKT3 therapy is effective but has several undesirableside effects; its use results in the release of numerous cytokinesincluding tumor necrosis factor-α, interferon-γ, interleukin-2, andinterleukin-6, resulting in fever, chills and gastrointestinal distress(for a review see Parlevliet et al., Transplant Intl. 5: 234-246 (1992);Dantal et al., Curr. Opin. Immunol. 3: 740-747 (1991)). Cyclosporin A iseffective but also has serious side effects (for a review see Barry,Drugs, 44: 554-566 (1992)).

SUMMARY OF THE INVENTION

The instant invention provides humanized anti-CD11a antibodies.Preferred antibodies bind to the I-domain of human CD11a (e.g. to"epitope MHM24" as herein defined) and/or bind CD11a with an affinity ofabout 1×10⁻⁸ M or stronger. In preferred embodiments, the antibody hasan IC50 (nM) value of no more than about 1 nM for preventing adhesion ofJurkat cells to normal human epidermal keratinocytes expressing ICAM-1.Preferred humanized antibodies are those which have an IC50 (nM) valueof no more than about 1 nM in the mixed lymphocyte response (MLR) assay.This IC50 for a humanized antibody in the MLR assay is significantlybetter than that that for murine MAb 25.3, which has been previouslytested in vivo (Fischer et al., Blood 77: 249-256 (1991); Stoppa et al.,Transplant Intl. 4: 3-7 (1991); Hourmant et al., Transplantation 58:377-380 (1994)).

The humanized anti-CD11a antibody may have a heavy chain variable regioncomprising the amino acid sequence of CDR1 (GYSFTGHWMN; SEQ ID NO:10)and/or CDR2 (MIHPSDSETRYNQKFKD; SEQ ID NO:11) and/or CDR3 (GIYFYGTTYFDY;SEQ ID NO:12) of humanized antibody MHM24 F(ab)-8 in FIG. 1 and/or alight chain variable region comprising the amino acid sequence of CDRI(RASKTISKYLA; SEQ ID NO:13) and/or CDR2 (SGSTLQS; SEQ ID NO:14) and/orCDR3 (QQHNEYPLT; SEQ ID NO:15) of humanized antibody MHM24 F(ab)-8 inFIG. 1. In other embodiments, the antibody comprises an amino acidsequence variant of one or more of the CDRs of humanized MHM24 antibodyF(ab)-8, which variant comprises one or more amino acid insertion(s)within or adjacent to a CDR residue and/or deletion(s) within oradjacent to a CDR residue and/or substitution(s) of CDR residue(s) (withsubstitution(s) being the preferred type of amino acid alteration forgenerating such variants). Such variants will normally having a bindingaffinity for human CD11a which is no more than about 1×10⁻⁸ M.

In preferred embodiments, the humanized antibody includes a light chainvariable region comprising the amino acid sequence of SEQ ID NO:2 and/ora heavy chain variable region comprising the amino acid sequence of SEQID NO:5 of humanized antibody MHM24 F(ab)-8 in FIG. 1 and/or amino acidsequence variants thereof.

As described herein, it has been possible to reengineer a humanizedantibody that bound human CD11a antigen, but not significantly to rhesusCD11a antigen, so as to confer an ability to bind to rhesus CD11a (i.e.a "rhesusized" antibody). In this embodiment, the antibody which bindsrhesus CD11a may, for example, comprise the the CDR2 amino acid sequencein SEQ ID NO:23. The other CDRs may be the same as those for humanizedMHM24 antibody F(ab)-8. Thus, the antibody may comprise the amino acidsequence of the "rhesusized" heavy chain in SEQ ID NO:24, optionallycombined with a light chain comprising the amino acid sequence in SEQ IDNO:2.

Various forms of the antibody are contemplated herein. For example, theanti-CD11a antibody may be a full length antibody (e.g. having a humanimmunoglobulin constant region) or an antibody fragment (e.g. aF(ab')₂). Furthermore, the antibody may be labeled with a detectablelabel, immobilized on a solid phase and/or conjugated with aheterologous compound (such as a cytotoxic agent).

Diagnostic and therapeutic uses for the antibody are contemplated. Inone diagnostic application, the invention provides a method fordetermining the presence of CD11a protein comprising exposing a samplesuspected of containing the CD11a protein to the anti-CD11 a antibodyand determining binding of the antibody to the sample. For this use, theinvention provides a kit comprising the antibody and instructions forusing the antibody to detect the CD11a protein.

The invention further provides: isolated nucleic acid encoding theantibody; a vector comprising that nucleic acid, optionally operablylinked to control sequences recognized by a host cell transformed withthe vector; a host cell comprising that vector; a process for producingthe antibody comprising culturing the host cell so that the nucleic acidis expressed and, optionally, recovering the antibody from the host cellculture (e.g. from the host cell culture medium).

The invention also provides a composition comprising the humanizedanti-CD11a antibody and a pharmaceutically acceptable carrier ordiluent. This composition for therapeutic use is sterile and may belyophilized. The invention further provides a method for treating amammal suffering from a LFA-1 mediated disorder, comprisingadministering a pharmaceutically effective amount of the humanizedanti-CD11a antibody to the mammal. For such therapeutic uses, otherimmunosuppressive agents or adhesion molecule antagonists (e.g. anotherLFA-1 antagonist or a VLA-4 antagonist) may be co-administered to themammal either before, after, or simultaneously with, the humanizedanti-CD11a antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the amino acid sequences of murine MHM24 light chain (SEQID NO: 1), humanized MHM24 F(ab)-8 light chain (SEQ ID NO:2), humanconsensus sequences of light chain subgroup KI (humKI) (SEQ ID NO:3).

FIG. 1B shows the amino acid sequences of murine MHM24 heavy chain (SEQID NO:4), humanized MHM24 F(ab)-8 heavy chain(SEQ ID NO:5), humanconsensus sequences of heavy chain subgroup III (humII) (SEQ ID NO:6)and "rhesusized" antibody mutant heavy chain of the Example (SEQ IDNO:24).

In FIGS. 1A and 1B, hypervariable regions based on sequencehypervariability (Kabat et al., Sequences of Proteins of ImmunologicalInterest. 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)) are endosed within brackets and hypervariableloops based on structure of F(ab)-antigen complexes (Chothia et al.,Nature 342: 8767 (1989)) are in italics. Residue numbering is accordingto Kabat et al., with insertions shown as a, b, and c.

FIG. 2 shows sequences of human CD11a I-domain (SEQ ID NO:7) and rhesusCD11a I-domain (SEQ ID NO:8). β-strands and α-helices are underlined andlabeled according to Qu et al., Proc. Natl. Acad. Sci. 92: 10277-10281(1995). The rhesus I-domain sequence (rhCD11a) shows only the fourdifferences from human I-domain. The binding epitope for the MHM24 MAb(SEQ ID NO:9) is shown in bold (Champe et al., J. Biol Chem. 270:1388-1394 (1995)).

FIG. 3 depicts inhibition of human Jurkat T-cells to normal humankeratinocytes by murine MHM24 (filled circles), chimeric MHM24 (opentriangles), humanized MHM24 (HulgG1) (filled squares), and a human IgG1isotype control (+). Percent binding measured by fluorescence of labeledJurkat cells.

FIGS. 4A-4C show inhibition of binding of rhesus lymphocytes to normalhuman keratinocytes (FIG. 4A), rhesus lymphocytes to recombinant humanICAM-1 coated on plates (FIG. 4B), and rhesus/human CD11achimera-transfected 293 cells to normal human keratinocytes (FIG. 4C).Inhibition by rhesus-binding MHM24 (RhlgG1) (filled squares), anti-CD18MHM23 (filled circles), a human IgG1 isotype control (+) (FIGS. 4A and4C), and a murine IgG1 isotype control (+) (FIG. 4B). Percent bindingmeasured by fluorescence of labeled lymphocytes (FIGS. 4A and B) orlabeled 293 cells (FIG. 4C).

FIG. 5 shows human mixed lymphocyte response assy (MLR) is blocked bymurine MHM24 (filled circles), humanized MHM24 (HulgG1) (filledsquares), and a humanized isotype IgG1 control (filled diamond). Percentstimulation index (%SI) is the ratio of the response at a given MAbconcentration to the maximal response with no MAb present. Data isrepresentative of multiple assays using at least two differentstimulator/responder pairs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

Unless indicated otherwise, the term "CD11a" when used herein refers tothe alpha subunit of LFA-1 from any mammal, but preferably from a human.The CD11a may be isolated from a natural source of the molecule or maybe produced by synthetic means (e.g., using recombinant DNA technology.)The amino acid sequence for human CD11a is described in EP 362 526B1,for example.

The term "I-domain" of CD11a refers to the region of this moleculedelineated in Champe et al., J. Biol. Chem. 270: 1388-1394 (1995) and/orQu et al. Proc. Natl. Acad. Sci. 92: 10277-10281 (1995). The amino acidsequences of human CD11a I-domain (SEQ ID NO:7) and rhesus CD11aI-domain (SEQ ID NO:8) are depicted in FIG. 2 herein.

The term "epitope MHM24" when used herein, unless indicated otherwise,refers to the region in the I-domain of human CD11a to which the MHM24antibody (see Example below) binds. This epitope comprises the aminoacid sequence of SEQ ID NO:9 and, optionally, other amino acid residuesof CD11a and/or CD18.

The term "LFA-1-mediated disorder" refers to a pathological state causedby cell adherence interactions involving the LFA-1 receptor onlymphocytes. Examples of such disorders include T cell inflammatoryresponses such as inflammatory skin diseases including psoriasis;responses associated with inflammatory bowel disease (such as Crohn'sdisease and ulcerative colitis); adult respiratory distress syndrome;dermatitis; meningitis; encephalitis; uveitis; allergic conditions suchas eczema and asthma; conditions involving infiltration of T cells andchronic inflammatory responses; skin hypersensitivity reactions(including poison ivy and poison oak); atherosclerosis; leukocyteadhesion deficiency; autoimmune diseases such as rheumatoid arthritis,systemic lupus erythematosus (SLE), diabetes mellitus, multiplesclerosis, Reynaud's syndrome, autoimmune thyroiditis, experimentalautoimmune encephalomyelitis, Sjorgen's syndrome, juvenile onsetdiabetes, and immune responses associated with delayed hypersensitivitymediated by cytokines and T-lymphocytes typically found in tuberculosis,sarcoidosis, polymyositis, granulomatosis and vasculitis; perniciousanemia; chronic obstructive pulmonary disease (COPD); bronchitis;insulinitis; rhinitis; urticaria; glomerulonephritis; diseases involvingleukocyte diapedesis; CNS inflammatory disorder; multiple organ injurysyndrome secondary to septicaemia or trauma; autoimmune hemolyticanemia; myethemia gravis; antigen-antibody complex mediated diseases;nephrotic syndrome; malignancies (e.g., B-cell malignancies such aschronic lymphocytic leukemia or hairy cell leukemia); all types oftransplantations, including graft vs. host or host vs. graft disease;HIV and rhinovirus infection; pulmonary fibrosis; invasion of tumorcells into secondary organs etc.

The term "immunosuppressive agent" as used herein for adjunct therapyrefers to substances that act to suppress or mask the immune system ofthe host into which the graft is being transplanted. This would includesubstances that suppress cytokine production, downregulate or suppressself-antigen expression, or mask the MHC antigens. Examples of suchagents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat.No. 4,665,077), azathioprine (or cyclophosphamide, if there is anadverse reaction to azathioprine); bromocryptine; glutaraldehyde (whichmasks the MHC antigens, as described in U.S. Pat. No. 4,120,649);anti-idiotypic antibodies for MHC antigens and MHC fragments;cyclosporin A; steroids such as glucocorticosteroids, e.g., prednisone,methylprednisolone, and dexamethasone; cytokine or cytokine receptorantagonists including anti-interferon-γ, -β, or -α antibodies;anti-tumor necrosis factor-α antibodies; anti-tumor necrosis factor-βantibodies; anti-interleukin-2 antibodies and anti-IL-2 receptorantibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin;pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies;soluble peptide containing a LFA-3 binding domain (WO 90/08187 publishedJul. 26, 1990); streptokinase; TGF-β; streptodomase; RNA or DNA from thehost; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor (U.S.Pat. No. 5,114,721); T-cell receptor fragments (Offner et al., Science251: 430-432 (1991); WO 90/11294; and WO 91/01133); and T cell receptorantibodies (EP 340,109) such as T10B9. These agents are administered atthe same time or at separate times from the CD11a antibody, and are usedat the same or lesser dosages than as set forth in the art. Thepreferred adjunct immunosuppressive agent will depend on many factors,including the type of disorder being treated including the type oftransplantation being performed, as well as the patient's history, but ageneral overall preference is that the agent be selected fromcyclosporin A, a glucocorticosteroid (most preferably prednisone ormethylprednisolone), OKT-3 monoclonal antibody, azathioprine,bromocryptine, heterologous anti-lymphocyte globulin, or a mixturethereof.

"Treatment" refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

"Mammal" for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

The term "graft" as used herein refers to biological material derivedfrom a donor for transplantation into a recipient. Grafts include suchdiverse material as, for example, isolated cells such as islet cells andneural-derived cells (e.g. schwann cells), tissue such as the amnioticmembrane of a newborn, bone marrow, hematopoietic precursor cells, andorgans such as skin, heart, liver, spleen, pancreas, thyroid lobe, lung,kidney, tubular organs (e.g., intestine, blood vessels, or esophagus),etc. The tubular organs can be used to replace damaged portions ofesophagus, blood vessels, or bile duct. The skin grafts can be used notonly for burns, but also as a dressing to damaged intestine or to closecertain defects such as diaphragmatic hernia. The graft is derived fromany mammalian source, including human, whether from cadavers or livingdonors. Preferably the graft is bone marrow or an organ such as heartand the donor of the graft and the host are matched for HLA class IIantigens.

The term "donor" as used herein refers to the mammalian species, dead oralive, from which the graft is derived. Preferably, the donor is human.Human donors are preferably volunteer blood-related donors that arenormal on physical examination and of the same major ABO blood group,because crossing major blood group barriers possibly prejudices survivalof the allograft. It is, however, possible to transplant, for example, akidney of a type O donor into an A, B or AB recipient.

The term "transplant" and variations thereof refers to the insertion ofa graft into a host, whether the transplantation is syngeneic (where thedonor and recipient are genetically identical), allogeneic (where thedonor and recipient are of different genetic origins but of the samespecies), or xenogeneic (where the donor and recipient are fromdifferent species). Thus, in a typical scenario, the host is human andthe graft is an isograft, derived from a human of the same or differentgenetic origins. In another scenario, the graft is derived from aspecies different from that into which it is transplanted, such as ababoon heart transplanted into a human recipient host, and includinganimals from phylogenically widely separated species, for example, a pigheart valve, or animal beta islet cells or neuronal cells transplantedinto a human host.

"Increasing tolerance of a transplanted graft" by a host refers toprolonging the survival of a graft in a host in which it istransplanted, i.e., suppressing the immune system of the host so that itwill better tolerate a foreign transplant.

"Intermittent" or "periodic" dosing is a dosing that is continuous for acertain period of time and is at regular intervals that are preferablyseparated by more than one day.

"Selective tolerance" of the disorder refers to a tolerance by thehost's immune system for the specific agent causing the disorder, butretaining the ability of the host to reject a second allogeneic orxenogeneic graft. Preferably, the tolerance is such that the immunesystem is left otherwise intact.

The term "LFA-1 antagonist" refers to a molecule that acts as acompetitive inhibitor of the LFA-1 interaction with ICAM-1. Examples ofsuch molecules include antibodies directed against either CD11a (e.g.,the humanized anti-CD11 a antibodies described herein) or CD18 or both,antibodies to ICAM-1, and other molecules such as peptides (e.g.,peptidomimetic antagonists).

The term "VLA-4 antagonist" refers to a molecule that acts as acompetitive inhibitor of the VLA-4 interaction with VCAM. Examples ofsuch molecules include antibodies directed against either VLA-4 or VCAMand other molecules (e.g., peptidomimetic antagonists).

The term "antibody" is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity.

"Antibody fragments" comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab', F(ab')₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier "monoclonal" indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256: 495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol.Biol. 222: 581-597 (1991), for example.

The monoclonal antibodies herein specifically include "chimeric"antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad Sci. USA 81: 6851-6855 (1984)).

The term "hypervariable region" when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a"complementarity determining region" or "CDR" (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a "hypervariable loop" (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). "Framework"or "FR" residues are those variable domain residues other than thehypervariable region residues as herein defined.

"Humanized" forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321: 522-525 (1986); Reichmannet al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol.2: 593-596 (1992).

"Single-chain Fv" or "sFv" antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term "diabodies" refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) -V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

The expression "linear antibodies" when used throughout this applicationrefers to the antibodies described in Zapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fdsegments (V_(H) -C_(H) 1-V_(H) -C_(H) 1) which form a pair of antigenbinding regions. Linear antibodies can be bispecific or monospecific.

An "isolated" antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The term "epitope tagged" when used herein refers to the anti-CD 11aantibody fused to an "epitope tag". The epitope tag polypeptide hasenough residues to provide an epitope against which an antibodythereagainst can be made, yet is short enough such that it does notinterfere with activity of the CD11a antibody. The epitope tagpreferably is sufficiently unique so that the antibody thereagainst doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least 6 amino acid residues and usuallybetween about 8-50 amino acid residues (preferably between about 9-30residues). Examples include the flu HA tag polypeptide and its antibody12CA5 (Field et al. Mol. Cell. Biol. 8: 2159-2165 (1988)); the c-myc tagand the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al.,Mol. Cell. Biol. 5(12):3610-3616 (1985)); and the Herpes Simplex virusglycoprotein D (gD) tag and its antibody (Paborsky et al., ProteinEngineering 3(6): 547-553 (1990)). In certain embodiments, the epitopetag is a "salvage receptor binding epitope". As used herein, the term"salvage receptor binding epitope" refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule.

The term "cytotoxic agent" as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A "chemotherapeutic agent" is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeAdriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside ("Ara-C"),Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin,Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine,Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin,Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see U.S. Pat. No.4,675,187), Melphalan and other related nitrogen mustards.

The term "prodrug" as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy" BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., "Prodrugs: A Chemical Approach to Targeted DrugDelivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

The word "label" when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibody.The label may itself be detectable by itself (e.g., radioisotope labelsor fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

By "solid phase" is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g. an affinity chromatography column). This term also includesa discontinuous solid phase of discrete particles, such as thosedescribed in U.S. Pat. No. 4,275,149.

A "liposome" is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the anti-CD11a antibodies disclosed herein and, optionally, achemotherapeutic agent) to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

An "isolated" nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The expression "control sequences" refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, "operably linked"means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions "cell," "cell line," and "cell culture"are used interchangeably and all such designations include progeny.Thus, the words "transformants" and "transformed cells" indude theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

II. Modes for Carrying out the Invention

A. Antibody Preparation

A method for humanizing a nonhuman CD11a antibody is described in theExample below. In order to humanize an anti-CD11a antibody, the nonhumanantibody starting material is prepared. Exemplary techniques forgenerating such antibodies will be described in the following sections.

(i) Antigen preparation.

The CD11a antigen to be used for production of antibodies may be, e.g.,a soluble form of the extracellular domain of CD11a or other fragment ofCD11a (e.g. a CD11a fragment comprising the "MHM24 epitope", such asCD11a I-domain fragment). Alternatively, cells expressing CD11a at theircell surface can be used to generate antibodies. Such cells can betransformed to express CD11a and, optionally, CD18 or may be othernaturally occurring cells (e.g. human lymphoblastoid cells, see Hildrethet al. Eur. J. Immunol. 13: 202-208 (1983)) or Jurkat cells (see Examplebelow). Other forms of CD11a useful for generating antibodies will beapparent to those skilled in the art.

(ii) Polyclonal antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹ N═C═NR,where R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with 1/5 to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(iii) Monoclonal antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256: 495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOP-21 and M.C.-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107: 220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Recombinant production of antibodies will be described in moredetail below.

(iv) Humanization and amino acid sequence variants

The Example below describes a procedure for humanization of ananti-CD11a antibody. In certain embodiments, it may be desirable togenerate amino acid sequence variants of the humanized antibody,particularly where these improve the binding affinity or otherbiological properties of the humanized antibody.

Amino acid sequence variants of humanized anti-CD11a antibody areprepared by introducing appropriate nucleotide changes into thehumanized anti-CD11a antibody DNA, or by peptide synthesis. Suchvariants include, for example, deletions from, and/or insertions intoand/or substitutions of, residues within the amino acid sequences shownfor the humanized anti-CD11a F(ab)-8 (e.g. as in SEQ ID NO's 2 & 5). Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the humanized anti-CD11a antibody, suchas changing the number or position of glycosylation sites.

A useful method for identification of certain residues or regions of thehumanized anti-CD11 a antibody polypeptide that are preferred locationsfor mutagenesis is called "alanine scanning mutagenesis," as describedby Cunningham and Wells Science, 244: 1081-1085 (1989). Here, a residueor group of target residues are identified (e.g., charged residues suchas arg, asp, his, lys, and glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine) to affectthe interaction of the amino acids with CD11a antigen. Those amino acidlocations demonstrating functional sensitivity to the substitutions thenare refined by introducing further or other variants at, or for, thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to analyze the performance ofa mutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed humanizedanti-CD11a antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includehumanized anti-CD11a antibody with an N-terminal methionyl residue orthe antibody fused to an epitope tag. Other insertional variants of thehumanized anti-CD11a antibody molecule include the fusion to the N- orC-terminus of humanized anti-CD11a antibody of an enzyme or apolypeptide which increases the serum half-life of the antibody (seebelow).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the humanizedanti-CD11a antibody molecule removed and a different residue inserted inits place. The sites of greatest interest for substitutional mutagenesisinclude the hypervariable loops, but FR alterations are alsocontemplated. Table IV in the Example below provides guidance as tohypervariable region residues which can be altered. Hypervariable regionresidues or FR residues involved in antigen binding are generallysubstituted in a relatively conservative manner. Such conservativesubstitutions are shown in Table I under the heading of "preferredsubstitutions". If such substitutions result in a change in biologicalactivity, then more substantial changes, denominated "exemplarysubstitutions" in Table I, or as further described below in reference toamino acid classes, are introduced and the products screened.

                  TABLE I                                                         ______________________________________                                        Original    Exemplary    Preferred                                                                      Residue Substitutions Substitutions                 ______________________________________                                        Ala (A)     val; leu; ile                                                                              val                                                    Arg (R) lys; gln; asn lys                                                     Asn (N) gln; his; lys; arg gln                                                Asp (D) glu glu                                                               Cys (C) ser ser                                                               Gln (Q) asn asn                                                               Glu (E) asp asp                                                               Gly (G) pro; ala ala                                                          His (H) asn; gln; lys; arg arg                                                Ile (I) leu; val; met; ala; leu                                                phe; norleucine                                                              Leu (L) norleucine; ile; val; ile                                              met; ala; phe                                                                Lys (K) arg; gln; asn arg                                                     Met (M) leu; phe; ile leu                                                     Phe (F) leu; val; ile; ala; tyr leu                                           Pro (P) ala ala                                                               Ser (S) thr thr                                                               Thr (T) ser ser                                                               Trp (W) tyr; phe tyr                                                          Tyr (Y) trp; phe; thr; ser phe                                                Val (V) ile; leu; met; phe; leu                                                ala; norleucine                                                            ______________________________________                                    

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the humanized anti-CD11a antibody also may be substituted, generallywith serine, to improve the oxidative stability of the molecule andprevent aberrant crosslinking. Conversely, cysteine bond(s) may be addedto the antibody to improve its stability (particularly where theantibody is an antibody fragment such as an Fv fragment).

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants ofhumanized anti-CD11a antibody are prepared by a variety of methods knownin the art. These methods include, but are not limited to, isolationfrom a natural source (in the case of naturally occurring amino acidsequence variants) or preparation by oligonucleotide-mediated (orsite-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis ofan earlier prepared variant or a non-variant version of humanizedanti-CD11a antibody.

Ordinarily, amino acid sequence variants of the humanized anti-CD11aantibody will have an amino acid sequence having at least 75% amino acidsequence identity with the original humanized antibody amino acidsequences of either the heavy or the light chain (e.g. as in SEQ ID NO:2or 5), more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, and most preferably at least 95%. Identity orhomology with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the humanized anti-CD11a residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions (as defined in Table I above) as part of the sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the antibody sequence shall be construedas affecting sequence identity or homology.

(v) Screening for biological properties

Antibodies having the characteristics identified herein as beingdesirable in a humanized anti-CD11a antibody are screened for.

To screen for antibodies which bind to the epitope on CD11a bound by anantibody of interest (e.g., those which block binding of the MHM24antibody to CD11a ), a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al., J.Biol. Chem. 270: 1388-1394 (1995), can be performed to determine whetherthe antibody binds an epitope of interest.

Antibody affinities (e.g. for human CD11a or rhesus CD11a) may bedetermined by saturation binding using either peripheral bloodmononuclear cells or rhesus leukocytes as described in the Examplebelow. According to this method for determining antibody affinity,lymphocytes or rhesus leukocytes are added to the plates in a volume of170 μl per well and plates are incubated for 2 hr at room. Afterincubation, cells are harvested and washed 10 times. Samples are thencounted. Data is transformed from counts per minute to nanomolarity andfour-parameter curve-fitting of saturation plots (bound versus total)are then performed to determine K_(d) (app) values. Preferred humanizedantibodies are those which bind human CD11a with a K_(d) value of nomore than about 1×10⁻⁷ ; preferably no more than about 1×10⁸ ; morepreferably no more than about 1×10⁻⁹ ; and most preferably no more thanabout 2×10⁻¹⁰.

It is also desirable to select humanized antibodies which havebeneficial anti-adhesion properties in the "keratinocyte monolayeradhesion assay". Preferred antibodies are those which have an IC50 (nM)value of no more than about 250 nM; preferably no more than about 100nM; more preferably no more than about 1 nM and most preferably no morethan about 0.5 nM for preventing adhesion of Jurkat cells to normalhuman epidermal keratinocytes expressing ICAM-1. According to thisassay, normal human epidermal keratinocytes are removed from cultureflasks and resuspended in lymphocyte assay medium at a concentration of5×10⁵ viable cells/ml. Aliquots of 0.1 ml/well are then culturedovernight in flat-bottom 96-well plates; appropriate wells arestimulated by addition of interferon-gamma at 100 units/well. Jurkatclone E6-1 cells are labeled, washed, resuspended to 1×10⁶ cells/ml, andincubated with 2-fold serial dilutions starting at 500 ng/ml antibody at4° C. for 30 min. After removal of medium from the keratinocytemonolayer, 0.1 ml/well of labeled cells are added and incubated at 37°C. for 1 h. The wells are washed to remove non-attached cells andfluorescence is measured.

Desirable humanized anti-CDIIa antibodies are those which have an IC50(nM) value of no more than about 100 nM; preferably no more than about50 nM; more preferably no more than about 5 nM and most preferably nomore than about 1 nM in the mixed lymphocyte response (MLR) assay, usinghuman lymphocytes. For both human and rhesus MLR, peripheral bloodlymphocytes from two unrelated donors are isolated from whole,heparinized blood and are resuspended to a concentration of 3×10⁶cells/ml in RPMI 1640 (GIBCO) with additives as described in the Examplebelow. The stimulator cells are made unresponsive by irradiation.Responder cells at a concentration of 1.5×10⁵ cells per well areco-cultured with an equal number of stimulator cells in 96-well,flat-bottom plates. Two-fold serial dilutions of antibody starting at aconcentration of 10 nM are added to the cultures to give a total volumeof 200 μl/well. The cultures are incubated at 37° C. in 5% CO₂ for 5days and then pulsed with 1 μCI/well of [³ H]thymidine for 16 h and [³H]thymidine incorporation is measured.

(vi) Antibody fragments

In certain embodiments, the humanized CD11a antibody is an antibodyfragment. Various techniques have been developed for the production ofantibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,Joumal of Biochemical and Biophysical Methods 24: 107-117 (1992) andBrennan et al., Science 229: 81 (1985)). However, these fragments cannow be produced directly by recombinant host cells. For example, Fab'-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab')₂ fragments (Carter et al., Bio/Technology 10: 163-167(1992)). According to another approach, F(ab')₂ fragments can beisolated directly from recombinant host cell culture. Other techniquesfor the production of antibody fragments will be apparent to the skilledpractitioner.

(vii) Multispecific antibodies

In some embodiments, it may be desirable to generate multispecific (e.g.bispecific) humanized CD11a antibodies having binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the CD11a protein. Alternatively, ananti-CD11a arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g., CD2 orCD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcyRII(CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms tothe CD11a-expressing cell. Bispecific antibodies may also be used tolocalize cytotoxic agents to cells which express CD11a. These antibodiespossess an CD11a-binding arm and an arm which binds the cytotoxic agent(e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain,methotrexate or radioactive isotope hapten). Bispecific antibodies canbe prepared as full length antibodies or antibody fragments (e.g.,F(ab')₂ bispecific antibodies).

According to another approach for making bispecific antibodies, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H) 3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.,tyrosine or tryptophan). Compensatory "cavities" of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers. See WO96/27011 published Sep. 6, 1996.

Bispecific antibodies include cross-linked or "heteroconjugate"antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab')₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab' fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives isthen reconverted to the Fab'-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab'-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab'-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab')₂molecule. Each Fab' fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab' portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The"diabody" technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90: 6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V₄ domains of another fragment, thereby forming two antigen-bindingsites. Another strategy for making bispecific antibody fragments by theuse of single-chain Fv (sFv) dimers has also been reported. See Gruberet al., J. Immunol. 152: 5368 (1994). Alternatively, the bispecificantibody may be a "linear antibody" produced as described in Zapata etal. Protein Eng. 8(10): 1057-1062 (1995).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991).

(viii) Other modifications

Other modifications of the humanized anti-CD11a antibody arecontemplated. For example, it may be desirable to modify the antibody ofthe invention with respect to effector function, so as to enhance theeffectiveness of the antibody in treating cancer, for example. Forexample cysteine residue(s) may be introduced in the Fc region, therebyallowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated may have improved internalizationcapability and/or increased complement-mediated cell killing andantibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J.Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922(1992). Homodimeric antibodies with enhanced anti-tumor activity mayalso be prepared using heterobifunctional cross-linkers as described inWolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, anantibody can be engineered which has dual Fc regions and may therebyhave enhanced complement lysis and ADCC capabilities. See Stevenson etal., Anti-Cancer Drug Design 3: 219-230 (1989).

The invention also pertains to immunoconjugates comprising the antibodydescribed herein conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated anti-CD11a antibodies. Examples include ²¹² Bi, ¹³¹ I,¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a "receptor"(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a "ligand" (e.g., avidin) which is conjugatedto a cytotoxic agent (e.g., a radio nuclide).

The anti-CD11a antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab' fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.81(19): 1484 (1989)

The antibody of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as "abzymes", can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme conjugates can be prepared as describedherein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the anti-CD11aantibodies by techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature 312: 604-608 (1984)).

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. In this case, it may be desirable to modifythe antibody fragment in order to increase its serum half life. This maybe achieved, for example, by incorporation of a salvage receptor bindingepitope into the antibody fragment (e.g., by mutation of the appropriateregion in the antibody fragment or by incorporating the epitope into apeptide tag that is then fused to the antibody fragment at either end orin the middle, e.g., by DNA or peptide synthesis). See WO96/32478published Oct. 17, 1996.

The salvage receptor binding epitope generally constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or V_(H) region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C_(L) region or V_(L)region, or both, of the antibody fragment.

In one most preferred embodiment, the salvage receptor binding epitopecomprises the sequence (5' to 3'): PKNSSMISNTP (SEQ ID NO:16), andoptionally further comprises a sequence selected from the groupconsisting of HQSLGTQ (SEQ ID NO:17), HQNLSDGK (SEQ ID NO:18), HQNISDGK(SEQ ID NO:19), or VISSHLGQ (SEQ ID NO:20), particularly where theantibody fragment is a Fab or F(ab')₂. In another most preferredembodiment, the salvage receptor binding epitope is a polypeptidecontaining the sequence(s)(5' to 3'): HQNLSDGK (SEQ ID NO:18), HQNISDGK(SEQ ID NO:19), or VISSHLGQ (SEQ ID NO:20) and the sequence: PKNSSMISNTP(SEQ ID NO:16).

Covalent modifications of the humanized CD11a antibody are also includedwithin the scope of this invention. They may be made by chemicalsynthesis or by enzymatic or chemical cleavage of the antibody, ifapplicable. Other types of covalent modifications of the antibody areintroduced into the molecule by reacting targeted amino acid residues ofthe antibody with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵ I or ¹³¹ I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R-N═C═N-R'), where R and R' are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Hakimuddin, etal. Arch. Biochem. Biophys. 259: 52 (1987) and by Edge et al. Anal.Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moietieson antibodies can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. Meth. Enzymol. 138:350 (1987).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

B. Vectors, Host Cells and Recombinant Methods

The invention also provides isolated nucleic acid encoding the humanizedanti-CD11a antibody, vectors and host cells comprising the nucleic acid,and recombinant techniques for the production of the antibody.

For recombinant production of the antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.

(i) Signal sequence component

The anti-CD11a antibody of this invention may be produced recombinantlynot only directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native anti-CD11a antibody signal sequence,the signal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeastsecretion the native signal sequence may be substituted by, e.g., theyeast invertase leader, a factor leader (including Saccharomyces andKluyveromyces α-factor leaders), or acid phosphatase leader, the C.albicans glucoamylase leader, or the signal described in WO90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable.

The DNA for such precursor region is ligated in reading frame to DNAencoding the anti-CD11a antibody.

(ii) Origin of replication component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

(iii) Selection gene component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theanti-CD 11a antibody nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding anti-CD 11a antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3'-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85: 12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al, Bio/Technology, 9: 968-975(1991).

(iv) Promoter component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the anti-CD11aantibody nucleic acid. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter , β-lactamase and lactose promoter systems,alkaline phosphatase, a tryptophan (trp) promoter system, and hybridpromoters such as the tac promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems also willcontain a Shine-Dalgamo (S.D.) sequence operably linked to the DNAencoding the anti-CD11a antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3' end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3' end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Anti-CD11a antibody transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297: 598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

(v) Enhancer element component

Transcription of a DNA encoding the anti-CD11a antibody of thisinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297: 17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5' or 3' to theanti-CD11a antibody-encoding sequence, but is preferably located at asite 5' from the promoter.

(vi) Transcription termination component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5' and, occasionally 3', untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding anti-CD 11a antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

(vii) Selection and transformation of host cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-CD11 aantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger

Suitable host cells for the expression of glycosylated anti-CD11aantibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36: 59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y Acad. Sci.383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for anti-CD11a antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

(viii) Culturing the host cells

The host cells used to produce the anti-CD11a antibody of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma) are suitable for culturing the host cells. In addition, any ofthe media described in Ham et al., Meth. Enz. 58: 44 (1979), Barnes etal., Anal. Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704;4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195;or U.S. Pat. Re. No. 30,985 may be used as culture media for the hostcells. Any of these media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

(ix) Purification of anti-CD11a antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10: 163-167 (1992) describe a procedure forisolating antibodies which are secreted to the periplasmic space of E.coli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30min. Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human y1, y2, or y4 heavychains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein Gis recommended for all mouse isotypes and for human y3 (Guss et al.,EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H) 3 domain, the Bakerbond ABX™resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g.,from about 0-0.25M salt).

C. Pharmaceutical Formulations

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS--S bond formation through thio-disulfide interchange, stabilizationmay be achieved by modifying sulfhydryl residues, lyophilizing fromacidic solutions, controlling moisture content, using appropriateadditives, and developing specific polymer matrix compositions.

D. Non-therapeutic Uses for the Antibody

The antibodies of the invention may be used as affinity purificationagents. In this process, the antibodies are immobilized on a solid phasesuch a Sephadex resin or filter paper, using methods well known in theart. The immobilized antibody is contacted with a sample containing theCD11a protein (or fragment thereof) to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the CD11a protein, which is boundto the immobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0, that will release theCD11a protein from the antibody.

Anti-CD11a antibodies may also be useful in diagnostic assays for CD11aprotein, e.g., detecting its expression in specific cells, tissues, orserum.

For diagnostic applications, the antibody typically will be labeled witha detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

(a) Radioisotopes, such as ³⁵ S, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I. Theantibody can be labeled with the radioisotope using the techniquesdescribed in Current Protocols in Immunology, Volumes 1 and 2, Coligenet al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991) for exampleand radioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocydic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, N.Y., 73: 147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g., digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g., anti-digoxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

In another embodiment of the invention, the anti-CD11a antibody need notbe labeled, and the presence thereof can be detected using a labeledantibody which binds to the CD11a antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of CD11a protein in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radio nuclide (such as ¹¹¹ In,⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ¹²⁵ I, ³ H, ³² P or ³⁵ S) so that the tumor can belocalized using immunoscintiography.

E. Diagnostic Kits

As a matter of convenience, the antibody of the present invention can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labeled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied widelyto provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

F. Therapeutic Uses for the Antibody

It is contemplated that the anti-CD11a antibody of the present inventionmay be used to treat the various LFA-1 mediated disorders as describedherein.

The anti-CD11a antibody is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunosuppressive treatment,intralesional administration (including perfusing or otherwisecontacting the graft with the antibody before transplantation).Parenteral infusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, theanti-CD11a antibody is suitably administered by pulse infusion,particularly with declining doses of the antibody. Preferably the dosingis given by injections, most preferably intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g., 0.1-20mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays. An exemplary dosingregimen is disclosed in WO94/04188.

The antibody composition will be formulated, dosed, and administered ina fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The"therapeutically effective amount" of the antibody to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat the LFA-1-mediated disorder,including treating rheumatoid arthritis, reducing inflammatoryresponses, inducing tolerance of immunostimulants, preventing an immuneresponse that would result in rejection of a graft by a host orvice-versa, or prolonging survival of a transplanted graft. Such amountis preferably below the amount that is toxic to the host or renders thehost significantly more susceptible to infections.

The antibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Forexample, in rheumatoid arthritis, the antibody may be given inconjunction with a glucocorticosteroid. In addition, T cell receptorpeptide therapy is suitably an adjunct therapy to prevent clinical signsof autoimmune encephalomyelitis. For transplants, the antibody may beadministered concurrently with or separate from an immunosuppressiveagent as defined above, e.g., cyclosporin A, to modulate theimmunosuppressant effect. Alternatively, or in addition, VLA-4antagonists or other LFA-1 antagonists may be administered to the mammalsuffering from a LFA-1 mediated disorder. The effective amount of suchother agents depends on the amount of anti-CD11a antibody present in theformulation, the type of disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as used hereinbefore or about from 1 to 99% of theheretofore employed dosages.

G. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is the anti-CD11a antibody.The label on, or associated with, the container indicates that thecomposition is used for treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

EXAMPLE Production of Humanized Anti-CD11a Antibodies

This example describes the humanization and in vitro biological efficacyof a murine anti-human CD11a monoclonal antibody, MHM24 (Hildreth etal., Eur. J. Immunol. 13: 202-208 (1983)). Previous studies on themurine MHM24 have shown that it, like other anti-CD11a antibodies, caninhibit T cell function (Hildreth et al., J. Immunol. 134: 3272-3280(1985); Dougherty et al., Eur. J. Immunol. 17: 943-947 (1987)). Both themurine and humanized MAbs effectively prevent adhesion of human T cellsto human keratinocytes and the proliferation of T cells in response tononautologous leukocytes in the mixed lymphocytes response (MLR), amodel for responsiveness to MHC class II antigens (McCabe et al.,Cellular Immunol. 150: 364-375 (1993)). However, both the murine(Reimann et al., Cytometry, 17: 102-108 (1994)) and humanized MAbs didnot cross-react with nonhuman primate CD11a other than chimpanzee CD11a.In order to have a humanized MAb available for preclinical studies inrhesus, the humanized MAb was re-engineered to bind to rhesus CD11a bychanging four residues in one of the complementarity-determiningregions, CDR-H2, in the variable heavy domain. Cloning and molecularmodeling of the rhesus CD11a I-domain suggested that a change from alysine residue in human CD11a I-domain to glutamic acid in rhesus CD11aI-domain is the reason that the murine and humanized MAbs do not bindrhesus CD11a.

Materials and Methods

(a) Construction of humanized F(ab')s

The murine anti-human CD11a MAb, MHM24 (Hildreth et al., Eur J. Immunol.13: 202-208 (1983); Hildreth et al., J Immunol. 134: 3272-3280 (1985)),was cloned and sequenced. In order to have a plasmid useful formutagenesis as well as for expression of F(ab)s in E. coli, the phagemidpEMX1 was constructed. Based on the phagemid pb0720, a derivative ofpB0475 (Cunningham et al., Science 243: 1330-1336 (1989)), pEMX1contains a DNA fragment encoding a humanized K-subgroup I light chainand a humanized subgroup III heavy chain (VH-CH1) and an alkalinephosphatase promotor and Shine-Dalgamo sequence both derived fromanother previously described pUC119-based plasmid, pAK2 (Carter et al.,Proc. Natl. Acad. Sci. USA 89: 4285 (1992)). A unique Spel restrictionsite was also introduced between the DNA encoding for the F(ab) lightand heavy chains.

To construct the first F(ab) variant of humanized MHM24, F(ab)-1,site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82: 488(1985)) was performed on a deoxyuridine-containing template of pEMX1;all six CDRs were changed to the MHM24 sequence. All other F(ab)variants were constructed from a template of F(ab)-1. Plasmids weretransformed into E. coli strain XL-1 Blue (Stratagene, San Diego,Calif.) for preparation of double-and single-stranded DNA. For eachvariant both light and heavy chains were completely sequenced using thedideoxynucleotide method (Sequenase, U.S. Biochemical Corp.). Plasmidswere transformed into E. coli strain 16C9, a derivative of MM294, platedonto LB plates containing 5 μg/ml carbenicillin, and a single colonyselected for protein expression. The single colony was grown in 5 mlLB-100 μg/ml carbenicillin for 5-8 h at 37° C. The 5 ml culture wasadded to 500 ml AP5-100 μg/ml carbenicillin and allowed to grow for 16 hin a 4 L baffled shake flask at 37° C. APS media consists of: 1.5 gglucose, 11.0 Hycase SF, 0.6 g yeast extract (certified), 0.19 g MgSO4(anhydrous), 1.07 g NH4Cl, 3.73 g KCl, 1.2 g NaCl, 120 ml 1 Mtriethanolamine, pH 7.4, to 1 L water and then sterile filtered through0.1 μm Sealkeen filter.

Cells were harvested by centrifugation in a 1 L centrifuge bottle(Nalgene) at 3000×g and the supematant removed. After freezing for 1 h,the pellet was resuspended in 25 ml cold 10 mM MES-10 mM EDTA, pH 5.0(buffer A). 250 μl of 0.1 M PMSF (Sigma) was added to inhibitproteolysis and 3.5 ml of stock 10 mg/ml hen egg white lysozyme (Sigma)was added to aid lysis of the bacterial cell wall. After gentle shakingon ice for 1 h, the sample was centrifuged at 40,000×g for 15 min. Thesupernatant was brought to 50 ml with buffer A and loaded onto a 2 mlDEAE column equilibrated with buffer A. The flow-through was thenapplied to a protein G-Sepharose CL-4B (Pharmacia) column (0.5 ml bedvolume) equilibrated with buffer A. The column was washed with 10 mlbuffer A and eluted with 3 ml 0.3 M glycine, pH 3.0, into 1.25 ml 1 MTris, pH 8.0. The F(ab) was then buffer exchanged into PBS using aCentricon-30 (Amicon) and concentrated to a final volume of 0.5 ml.SDS-PAGE gels of all F(ab)s were run to ascertain purity and themolecular weight of each variant was verified by electrospray massspectrometry.

(b) Construction of chimeric and humanized IgG

For generation of human IgG1 variants of chimeric (chlgG1) and humanized(HulgG1) MHM24, the appropriate murine or humanized variable light andvariable heavy (F(ab)-8, Table II) domains were subcloned into separatepreviously described pRK vectors (Gorman et al., DNA Protein Eng. Tech.2: 3 (1990)). Alanine-scan variants were constructed by site-directedmutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82: 488 (1985)) of theHulgG1 light and heavy chain plasmids. The DNA sequence of each variantwas verified by dideoxynucleotide sequencing.

Heavy and light chain plasmids were cotransfected into anadenovirus-transformed human embryonic kidney cell line, 293 (Graham etal, J. Gen. Virol. 36: 59 (1977)), using a high efficiency procedure(Graham et al., J. Gen. Virol. 36:59 (1977); Gorman et al, Science, 221:551 (1983)). Media was changed to serum-free and harvested daily for upto 5 days. Antibodies were purified from the pooled supematants usingprotein A-Sepharose CL-4B (Pharmacia). The eluted antibody was bufferexchanged into PBS using a Centricon-30 (Amicon), concentrated to 0.5ml, sterile filtered using a Millex-GV (Millipore) and stored at 4° C.

The concentration of antibody was determined using total Ig-bindingELISA. The concentration of the reference humanized anti-p185^(HER) IgG1(Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992)) wasdetermined by amino acid composition analysis. Each well of a 96-wellplate was coated with 1 μg/ml of goat anti-human IgG F(ab')₂ (CappelLaboratories, Westchester, Pa.) for 24 h at 4° C. Purified antibodieswere diluted and added in duplicate to the coated plates. After 1.5 hincubation, the plates were washed with PBS-0.02% Tween 20 and 0.1 ml ofa 1:2000 dilution of horseradish peroxidase-conjugated sheep anti-humanIgG F(ab')₂ (Cappel) was added. After 1.5 h incubation the plates werewashed and 0.1 ml 0.2 mg/ml o-phenylenediamine dihydrochloride-0.01%hydrogen peroxide-PBS was added. After 10 min. the reaction was stoppedwith 2 M sulfuric acid and the O.D. 490 nm was read.

(c) Cloning of rhesus CD11a I-domain

The DNA sequence of the rhesus I-domain was obtained using RT-PCR andprimers derived from the human CD11a DNA sequence. Briefly, mRNA wasisolated from ˜10⁷ rhesus leukocytes using the Fast Track mRNApurification kit (Invitrogen). 10 μg mRNA was reverse transcribed usingMuLV reverse transcriptase. The first strand cDNA was then amplified by40 cycles of PCR using the primers:

5'-CACTTTGGATACCGCGTCCTGCAGGT-3' (forward) (SEQ ID NO:21) and

5'-CATCCTGCAGGTCTGCCTTCAGGTCA-3' (reverse) (SEQ ID NO:22).

A single band of the predicted size was purified from the PCR reactionusing agarose gel electrophoresis. The PCR product was digested withrestriction enzyme Sse83871 (Takara) and ligated to a humanCD11a-containing plasmid digested with the same restriction enzyme.There are two Sse83871 sites in the human CD11a sequence, one on eitherside of the I-domain. The resulting plasmid encoded a chimera consistingof human CD11a with a rhesus I-domain substituted for the humanI-domain. DNA sequence analysis revealed five amino acid differencesbetween human and rhesus. One difference was in the region N-terminal tothe I-domain (Thr59Ser) and the other four differences were in theI-domain itself: Val133lle, Arg189Gln, Lys197Glu, and Val308Ala (FIG.2).

(d) FACScan analysis of F(ab) and IgG binding to Jurkat cells

Aliquots of 10⁶ Jurkat T-cells were incubated with serial dilutions ofhumanized and control antibodies in PBS-0.1% BSA-10 mM sodium azidefor45 min at 4° C. The cells were washed and then incubated influorescein-conjugated goat anti-human F(ab')₂ (Organon Teknika,Westchester, Pa.) for 45 min at 4° C. Cells were washed and analysed ona FACScan (Becton Dickinson, Mountain View, Calif.). 8×10³ cells wereacquired by list mode and gated by forward light scatter versus sidelight scatter, thereby excluding dead cells and debris.

(e) Saturation binding to determine apparent K_(d) s

Radiolabeled antibodies were prepared using Iodo-Gen (Pierce, Rockford,Ill.) according to the manufacturer's instructions. 50 μg of antibodyand 1 mCi¹²⁵ I (DuPont, Wilmington, Del.) were added to each tube andincubated for 15 min at 25° C. Radiolabeled proteins were purified fromthe remaining free ¹²⁵ I using PD-10 columns (Pharmacia, Uppsala,Sweden) equilibrated in Hank's Balanced Salt Solution (HBSS, LifeTechnologies, Grand Island, N.Y.) containing 0.2% gelatin.

Mononuclear cells were purified from heparinized human peripheral bloodcollected from two donors using Lymphocyte Separation Medium (LSM,Organon Teknika, Durham, N.C.) according to the manufacturer'sinstructions. The blood was centrifuged at 400×g for 40 min at 25° C.with no braking. Cells at the interface of the LSM and plasma wereharvested and then resuspended in HBSS-0.2% gelatin.

Leukocytes were purified from heparinized rhesus monkey peripheral bloodcollected from two individuals by Dextran sedimentation. Blood wasdiluted with an equal volume of 3% Dextran T500 (Pharmacia) in PBS andwas allowed to sediment undisturbed at 25° C. for 30 min. Aftersedimentation, cells remaining in suspension were harvested and pelletedby centrifugation at 400×g for 5 min. Residual erythrocytes were removedby two cycles of hypotonic lysis using distilled water and 2× HBSS.After erythrocyte lysis, cells were washed in PBS and then resuspendedin HBSS-0.2% gelatin.

Antibody affinities were determined by saturation binding using eitherperipheral blood mononuclear cells (murine MHM24 and HulgG1) or rhesusleukocytes (MHM23, RhlgG1). In each assay, a radiolabeled antibody wasserially diluted in HBSS-0.2% gelatin in quadruplicate. Nonspecificbinding was determined by the addition of 500nM final concentration ofhomologous unlabeled antibody in duplicate through the serial dilution.Human lymphocytes or rhesus leukocytes were added to the plates in avolume of 170 μl per well. Plates were incubated for 2 hr at roomtemperature on an orbital plate shaker. After incubation, cells wereharvested using a SKATRON™ cell harvester (Lier, Norway) and washed 10times with PBS containing 0.25% gelatin and 0.1% sodium azide. Sampleswere then counted for 1 min in an LBK. Wallac GammaMaster gamma counter(Gaithersburg, Md.). Data was transformed from counts per minute tonanomolarity and four-parameter curve-fitting of saturation plots (boundversus total) was then performed to determine K_(d) (app) values.

(f) Keratinocyte monolayer adhesion assay

Normal human epidermal keratinocytes (Clonetics, San Diego, Calif.) wereremoved from culture flasks with trypsin-EDTA, centrifuged, andresuspended in lymphocyte assay medium (RPMI 1640 (GIBCO)-10% fetal calfserum-1% penicillin/streptomycin) at a concentration of 5 ×10⁵ viablecells/ml. Aliquots of 0.1 ml/well were then cultured overnight inflat-bottom 96-well plates; appropriate wells were stimulated byaddition of interferon-gamma (Genentech, South San Francisco, Calif.) at100 units/well.

Jurkat clone E6-1 cells (ATCC, Rockville, Md.) or purified rhesuslymphocytes (see MLR methods) were labeled with 20 μg/ml Calcein AM(Molecular Probes, Eugene, Oreg.) at 37° C. for 45 min. After washingthree times with lymphocyte assay medium, Jurkat or rhesus lymphocytecells were resuspended to 1×10⁶ cells/ml and incubated withserially-diluted antibody at 4° C. for 30 min. After removal of mediumfrom the keratinocyte monolayer, 0.1 ml/well of labeled cells were addedand incubated at 37° C. for 1 h. The wells were washed five times with0.2 ml/well/wash of 37° C. lymphocyte medium to remove non-attachedcells. Fluorescence was measured using a Cytofluor 2300 (Millipore,Bedford, Mass.).

A rhesus-human chimeric CD11a (Rh/HuCD11a) comprising human CD11a with arhesus I-domain was constructed by site-directed mutagenesis (Kunkel,Proc. Natl. Acad. Sci. USA 82: 488 (1985)) on a deoxyuridine-containingtemplate plasmid encoding human CD11a. Four residues were altered:Val133lle, Arg189Gln, Lys197Glu, and Val308Ala (FIG. 2). Plasmids codingfor Rh/HuCD11a and human CD11b (EP 364, 690) were cotransfected into anadenovirus-transformed human embryonic kidney cell line, 293 (Graham etal., J Gen. Virol. 36: 59 (1977)), using a high efficiency procedure(Graham et al., J Gen. Virol. 36: 59 (1977); Gorman et al., Science,221: 551(1983)). Rh/HuCD11a-transfected 293 cells were labeled with 20μg/ml Calcein AM at 37° C. for 45 min. After washing three times withlymphocyte assay medium, Rh/HuCD11a-transfected 293 cells wereresuspended to 1×10⁶ cells/ml and incubated with serially-dilutedantibody at 4° C. for 30 min. After removal of medium from thekeratinocyte monolayer, 0.1 ml/well of labeled 293 cells was added andincubated at 37° C. for 1 h. The wells were washed five times with 0.2ml/well/wash of 37° C. lymphocyte medium to remove non-attached cells.Fluorescence was measured using a Cytofluor 2300.

(g) ICAM adhesion assay

Maxisorp (Nunc) 96-well plates were coated with 0.1 ml/well of 1 μg/mlgoat anti-human IgG Fc (Caltag) for 1 h at 37° C. After washing theplates three times with PBS, the plates were blocked with 1% BSA-PBS for1 h at 25° C. The plates were then washed three times with PBS and 0.1ml/well of 50 ng/ml recombinant human ICAM-IgG was added and incubatedovernight.

The ICAM-IgG consisted of the five extracellular domains of human ICAMfused onto a human IgG Fc. A plasmid for the expression of a humanICAM-1 (Simmons et al. Nature 331: 624-627 (1988) and Staunton et al.Cell 52: 925-933 (1988)) immunoadhesin called pRK.5dICAMGaIg wasconstructed. It contains the five Ig-like domains of ICAM-1, a six aminoacid cleavage site recognized by an H64A variant of subtilisin BPN',Genenase I (Carter et al. Proteins: Structure, Function and Genetics 6:240-248 (1989)), and the Fc region from human IgG1 (Ellison et al.Nucleic Acids Research 10: 4071-4079 (1982)) in the pRK5 vector (Eatonet al. Biochemistry 25: 8343-8347 (1986)). Human embryonic kidney 293cells (Graham et al. J. Gen. Virol. 36: 59 (1977)) were stablytransfected with pRK.5dICAMGaIg and the RSV-neo plasmid (Gorman et al.Science 221: 551-553 (1983)) to generate a cell line expressing the fivedomain ICAM Ig (5dICAMIg). A clone was selected which expressed 20 μg/mlof secreted 5dICAMIg by enzyme-linked immunosorbent assay (ELISA), usingantibodies to human IgG Fc (Caltag, Burlingame, Calif.) and ICAM-1(BBIG-I1; R & D Systems, Minneapolis, Minn.). Cell culture supernatantfrom this cell line was loaded onto a Protein A column (ProsepA,Bioprocessing, Ltd., Durham, England) equilibrated in 0.01 M Hepesbuffer (pH 7.0), 0.15 M NaCI (HBS) and the column was washed with HBSfollowed by 0.01 M Hepes buffer (pH 7.0), 0.5 M NaCI, 0.5 M TMAC(tetra-methyl ammonium chloride) to remove non-specifically boundmaterial. The TMAC buffer was thoroughly washed from the column with HBSand the 5dICAMIg eluted with 0.01 M Hepes buffer (pH 7.0), 3.5 M MgCl2and 10% (w/v) glycerol. The protein A pool was dialyzed extensivelyagainst HBS and concentrated.

Purified rhesus lymphocytes (see MLR methods) were labeled with 20 μg/mlCalcein AM (Molecular Probes, Eugene, Oreg.) at 37° C. for 45 min. Afterwashing three times with lymphocyte assay medium, rhesus lymphocytecells were resuspended to 1×10⁶ cells/ml and incubated withserially-diluted antibody at 4° C. for 30 min. After removal of mediumfrom the ICAM-IgG coated plates, 0.1 ml/well of labeled cells were addedand incubated at 37° C. for 1 h. The wells were washed five times with0.2 ml/well/wash of 37° C. lymphocyte medium to remove non-attachedcells. Fluorescence was measured using a Cytofluor 2300 (Millipore,Bedford, Mass.).

(h) One-way mixed lymphocyte response (MLR)

For both human and rhesus MLR, peripheral blood lymphocytes from twounrelated donors were isolated from whole, heparinized blood usingLymphocyte Separation Medium (Organon Teknika, Durham, N.C.).Lymphocytes were resuspended to a concentration of 3×10⁶ cells/ml inRPMI 1640 (GIBCO)-10% human AB serum-1% glutamine-1%penicillin/streptomycin-1% non-essential amino acids-1% pyruvate-5×10⁻⁵M 2-β-mercaptoethanol-50 μg/ml gentamycin-5 μg/ml polymyxin B. Thestimulator cells were made unresponsive by irradiation with 3000 rads ina cesium irradiator. Responder cells at a concentration of 1.5×10⁵ cellsper well were co-cultured with an equal number of stimulator cells in96-well, flat-bottom plates. Serial two-fold dilutions of each antibodywere added to the cultures to give a total volume of 200 μl/well. Thecultures were incubated at 37° C. in 5% CO₂ for 5 days and then pulsedwith 1 μCi/well of [³ H] thymidine for 16 h. [³ H] thymidineincorporation was measured with a Beckman scintillation counter. Assayswere done in triplicate. A humanized anti-human p185^(HER2) MAb (Carteret al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992)) was used as isotypecontrol for HuIgG1 and RhIgG1. A murine anti-hamster tPA MAb (Genentech)was used as isotype control (murine IgG1) for the MHM23 MAb. MAb 25.3was purchased from Immunotech, Inc. (Westbrook, Me.).

(i) Computer graphics models of murine and humanized MHM24

Sequences of the VL and VH domains (FIGS. 1A & B) were used to constructa computer graphics model of the murine MHM24 VL-VH domains. This modelwas used to determine which framework residues should be incorporatedinto the humanized antibody. A model of F(ab)-8 was also constructed toverify correct selection of murine framework residues. Construction ofthe models was performed as described previously (Carter et al., Proc.Natl. Acad. Sci. USA 89: 4285 (1992); Eigenbrot et al., J. Mol. Biol.229: 969 (1993)).

Results

(a) Humanization

The consensus sequence for the human heavy chain subgroup III and thelight chain subgroup K | were used as the framework for the humanization(Kabat et al., Sequences of Proteins of Immunological Interest. 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) (FIGS. 1A & B). This framework has been successfully used in thehumanization of other murine antibodies (Carter et al., Proc. Natl.Acad. Sci. USA 89: 4285 (1992); Presta et al., J. Immunol. 151:2623-2632 (1993); Eigenbrot et al., Proteins 18: 49-62 (1994)). Allhumanized variants were initially made and screened for binding asF(ab)s expressed in E. Coli. Typical yields from 500 ml shake flaskswere 0.2-0.5 mg F(ab). Mass spectrometry verified the mass of each F(ab)to within 5 mass units.

CDR-H1 included residues H28-H35, which includes all exposed residuesfrom both Kabat et al., Sequences of Proteins of Immunological Interest.5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991) and Chothia et al. Nature 342: 877-883 (1989). The otherhypervariable loops were defined according to Chothia et al. (1989).Light chain residue numbers are prefixed with L; heavy chain residuenumbers are prefixed with H.

                  TABLE II                                                        ______________________________________                                        Binding of humanized MHM24 variants                                             to human CD11a on Jurkat cells                                                                               EC50 F(ab)/                                    Tem-   EC50 F(ab)-2.sup.b                                                   Variant                                                                              plate   Changes.sup.a                                                                           Purpose Mean   S. D.                                                                              N                                ______________________________________                                        F(ab)-1                                                                              Human   ArgH71Val Straight                                                                              no          2                                   FR  CDR swap binding                                                         F(ab)-2 F(ab)-1 AlaH60Asn Extended 1.0  4                                       AspH61Gln CDR-H2                                                              SerH62Lys (Kabat et                                                           ValH63Phe al. (1991))                                                         GlyH65Asp                                                                   F(ab)-3 F(ab)-2 PheH67Ala Packing; 1.2 0.33  3                                   CDR-H2                                                                     F(ab)-4 F(ab)-2 ValH71Arg Packing; 2.9  1                                        CDR-H1, H2                                                                 F(ab)-5 F(ab)-2 AsnH73Lys Framework 0.043 0.015 4                               LysH75Ser loop in VH                                                          AsnH76Ser                                                                   F(ab)-6 F(ab)-5 SerL53Thr Extended 0.012 0.005 4                                GluL55Gln CDR-L2                                                            F(ab)-7 F(ab)-6 PheH27Tyr Extended 0.004 0.002 4                                 CDR-H1                                                                     F(ab)-8 F(ab)-7 SerH75Lys Framework 0.004 0.002 4                               SerH76Asn loop in VH                                                        HuIgG1.sup.c   back, 0.004 0.002 4                                            chIgG1.sup.d   to hyman 0.006 0.005 4                                       ______________________________________                                         .sup.a Murine residues are in bold; residue numbers are according to Kaba     et aL, Sequences of Proteins of Immunological Interest 5th Ed. Public         Health Service, National Institutes of Health, Bethesda, MD (1991).           .sup.b Mean and standard deviation are the average of the ratios              calculated for each of the independent FACScan assays; the EC50 for F(ab)     was 771 ± 320 ng/ml.                                                       .sup.c HuIgG1 is F(ab)8 VL and VH domains fused to human constant light       and heavy chains.                                                             .sup.d chIgG1 is chimeric IgG with murine VL and VH domains fused to huma     constant light and heavy chains.                                         

In the initial variant F(ab)-1 the CDR residues were transferred fromthe murine antibody to the human framework. In addition, residue H71 waschanged from the human Arg to murine Val since this residue has beenshown previously to affect the conformations of CDR-H1 and CDR-H2(Chothia et al., Nature, 342: 877-883 (1989); Tramontano J. Mol. Biol.215: 175 (1990)). This F(ab) showed no detectable binding. In F(ab)-2CDR-H2 was extended to include the sequence-based definition (i.e.,including residues H60-H65). The EC50 for F(ab)-2 binding to human CD11awas 771±320 ng/ml, which was 148-fold weaker than the EC50 of thechimeric IgG1 (5.2±3.0 ng/ml).

In previous humanizations, it has been found that residues in aframework loop (FR-3) adjacent to CDR-H1 and CDR-H2 can affect binding(Eigenbrot et al., Proteins 18: 49-62 (1994)). In F(ab)-5 three residuesin this loop were changed to their murine counterpart and this variantshowed a 23-fold improvement in binding (Table II). Alteration of humanresidues at positions L53 and L55 to murine (i.e. SerL53Thr andGluL55GIn) further improved binding by another 4-fold (F(ab)-6, TableII); this effectively converted CDR-L2 from the structure-baseddefinition (residues L50-L52) to the sequence-based definition (residuesL50-L56). Subsequent alteration of PheH27 to murine Tyr in CDR-H1resulted in an additional 3-fold improvement (F(ab)-7; Table II).Finally, based on the models of murine and humanized MHM24, two of thethree murine residues (H75 and H76) in FR-3 were changed back to humanand it was found that these two residues had no effect on binding (cf.F(ab)-7 and F(ab)-8, Table II). The average EC50 for F(ab)-8 wasslightly better than that of the chimeric IgG1 (Table II). Not allchanges from human to murine resulted in improved binding. PheH67 waschanged to murine Ala since this position had been previously found toaffect binding (Presta et al., J. Immunol. 151: 2623-2632 (1993)) but noeffect was evident (F(ab)-3, Table II). Changing ValH71 back to thehuman Arg effected a 3-fold reduction in binding (F(ab)-4, Table II),supporting inclusion of ValH71 in F(ab)-1.

The VL and VH domains from F(ab)-8 were transferred to human IgGlconstant domains. The full length intact antibody, HulgG1, showed anEC50 equivalent to F(ab)-8 and improved compared to the full lengthchimeric IgG1 (Table II). When data for all assays of HulgG1 isconsidered, including its use as a standard for the alanine-scan and MLRassays (see below), the EC50 for HulgG1 against human CD11a was0.042±0.072 nM (N=15). Saturation binding analysis was also performed todetermine the apparent dissociation constants, K_(d) (app): 0.15±0.02 nMfor murine MHM24 and 0.15±0.04 nM for HulgG1 (Table III).

                  TABLE III                                                       ______________________________________                                        Apparent K.sub.d by saturation binding to                                       human lymphocytes and rhesus leukocytes                                           muMHM24    HuIgG1     mUMHM23 RhIgG1                                      K.sub.d (app) k.sub.d (app) K.sub.d (app) K.sub.d (app)                       nM nM nM nM                                                                 ______________________________________                                        human 0.16 +/- 0.01                                                                            0.11 +/- 0.08                                                  donor 1                                                                       human 0.13 +/- 0.02 0.18 +/- 0.03                                             donor 2                                                                       rhesus   3.9 +/- 0.31 3.9 +/- 1.04                                            donor 1                                                                       rhesus   4.5 +/- 0.51 n.d.                                                    donor 2                                                                       rhesus    2.8 +/- 1.1.sup.a                                                   donor 3                                                                       rhesus    2.7 +/- 0.9                                                         donor 3                                                                     ______________________________________                                         *Assays of rhesus donor 3 were performed using two batches of RhIgG1; the     assays were performed in the presence of 1 mg/ml human IgG1 to block Fc       receptor interaction.                                                         .sup.(b) AlanineScan of CDR Residues                                     

To determine which CDR residues were involved in binding to human CD11aan alanine-scan (Cunningham et al, Science 244:1081 (1989)) wasperformed on the CDR residues of HulgG1. Each variant was tested forbinding to CD11a on Jurkat cells. In the light chain only CDR-L3contributes to binding. HisL91 had a large effect (Table IV) and isprobably conformational since this side chain should be partiallyburied. Residues AsnL92 and TyrL94 had more modest effect, reducingbinding by 3-fold and 12-fold, respectively. Note however thatsimultaneously changing these two residues to alanine (as well asGluL93Ala) had a non-additive effect on binding (variant L3, Table IV).

                                      TABLE IV                                    __________________________________________________________________________    Alanine-scan of humanized MHM24 CDR residues                                                             Human CD11a                                                                             Rhesus CD11a                                Var.EC50/HulgG1    Var.EC50/HulgG1                                           Variant IgG1         EC50.sup.b             EC50.sup.c                      CDR Sequence.sup.a         Mean                                                                              S.D.                                                                              N Mean                                                                              S.D.                                                                              N                                __________________________________________________________________________    CDR-H1                                                                              G Y S F T G HWMN                                                           - H1.sup.d         A   A   A                    5.9    0.8   2   nb                                                       - SerH28Ala       A                                                                             6.9                                                       0.1   2   nb                        - ThrH30Ala           A                        1.7    0.3   2   1.3                                                       - GlyH31Ala             A                                                                       1.2                                                       0.1   2   2.4                       - HisH32Ala               A                    2.3    0.2   2   nb                                                        - TrpH33Ala                                                                 A                >870                                                            1   nb                           - CDR-H2       M I H P S D S E T R Y N Q K F K D                              - H2               A   A   A                 14.1    7.8   10   0.055                                                   0.050   15                          - H2B                    A   A   A            >600           2   nb                                                       - H2A1             A   A                                                                      10.8                                                        7.3   6   0.013 0.012   10                                                      - H2A2                 A                                                    A                  1.9                                                        0.1   2   1.1   0.1      2                                                      - H2A3             A                                                        A                  4.6                                                        0.2   2   1.3                       - HisH52Ala        A                          1.5    0.2   2   0.7                                                        - HisH52Ser        S                                                                           5.0                                                        0.3   2   nb                        - SerH53Ala            A                      1.8   0.1   2   0.7                                                         - AspH54Ala              A                                                                    147    18                                                      2   0.3                          - SerH55Ala                A                  1.3    0.1   2   1.3                                                        - SerH55Asn                                                                 N                  2.1                                                        0.2   2   nb                        - SerH55Gln                Q                  2.9    1.4   2   6.7                                                        - GluH56Ala                                                                   A                4.0                                                        0.6   2   0.3                       - ArgH58Ala                      A             1.1    0.1   2   3.3                                                       - GlnH61Ala                                                                             A        4.1                                                      0.1   2   5.3                       - LysH62Ala                              A      1.8    0.2   2   4.9                                                      - LysH64Ala                                                                                   A   2.5                                                     0.1   2   1.2                       - AspH65Ala                                     A  0.8    0.1   2                                                       1.1                                 - FR-3                                                                        - LysH73Ala                                   5.2    0.9   2  nb                                                          - LysH73Arg                                                                                    4.6                                                        1.1   2    5.5                      - CDR-H3      G I Y F Y G T T Y F D                                           - H3              A   A                      >900           2   nb                                                        - H3B                                                                       A A A                 34.7                                                    13.6   2   nb                       - TyrH97Ala       A                         10.9    2.1   2   nb                                                          - TyrH99Ala           A                                                                        1.4   0.1                                                    2   nb                            - ThrH100aAla             A                    2.3    0.6   2   nb                                                        - ThrH100bAla                                                                A                   1.4                                                      0.2   2   nb                        - TyrH100cAla                 A                 7.6    1.0   2   nb                                                       - CDR-L1      R A S K T I                                                   S K Y L A                           - L1                A A   A A                   1.3    0.3   3    1.0                                                     - CDR-L2      S G S T L Q                                                   S                                   - L2          A   A A                         1.1    0.0   2   nb                                                         - SerL50Ala   A                                                                               1.2    0.5                                                    2    2.7                          - SerL52Ala       A                          1.1    0.2   2   13.3                                                        - ThrL53Ala         A                                                                          0.9                                                        0.4   2    0.7                      - CDR-L3      Q Q H N E Y P L T                                               - L3                A A A                      >900           2   nb                                                      - HisL91Ala       A                                                                          >900                                                            2   nb                           - AsnL92Ala         A                         3.3    0.7   2    3.3                                                       - GluL93Ala           A                                                                        1.7                                                        0.2   2    2.9                      - TyrL94Ala             A                     11.8    0.1   2   nb                                                        - hu4D5.sup.e                                                                                    >900                                                            5   nb                    __________________________________________________________________________     .sup.a CDRs and FR3 are as defined in Kabat et al., (1991) supra.             .sup.b EC50 HulgG1 for human CD11a = 0.042 nM (S.D. = 0.072; N = 15).         .sup.c EC50 HulgG1 for rhesus CD11a = 45.6 nM (S.D. = 40.4; N = 16); all      values for rhesus CD11a are for a single binding assay unless otherwise       noted; nb denotes binding of variant is greater than 10fold weaker than       HulgG1.                                                                       .sup.d Multiple alanine variants:                                             H1, SerH28Ala/ThrH30Ala/HisH32Ala;                                            H2, HisH52Ala/SerH53Ala/SerH55Ala;                                            H2B, AspH54Ala/GluH56Ala/ArgH58Ala;                                           H2A1, HisH52Ala/SerH53Ala;                                                    H2A2, SerH53Ala/SerH55Ala;                                                    H2A3, HisH52Ala/SerH55Ala;                                                    H3, TyrH97Ala/TyrH99Ala;                                                      H3B, ThrH100aAla/ThrH100bAla/TyrH100cAla;                                     L1, LysL27Ala/ThrL28Ala/SerL30Ala/LysL31Ala;                                  L2, SerL50Ala/SerL52Ala/ThrL53Ala;                                            L3 AsnL92Ala/GluL93Ala/TyrL94Ala.                                             .sup.e hu4D5 is a humanized antip185.sup.HER2 antibody with the same IgG1     framework as the huMHM24 antibody (Carter et al., Proc. Natl. Acad. Sci.      USA 89:4285 (1992)).                                                     

In the heavy chain, CDR-H2 and CDR-H3 are the prominent contributors tothe binding. CDR-H1 residue TrpH33Ala had a large effect but this ismost likely due to a conformational change as TrpH33 should be partiallyburied. The most important single residue contributing to the binding isAspH54 in CDR-H2; changing this residue to alanine effected a 147-foldreduction in binding (Table IV). Other residues in CDR-H2 involved inbinding include GluH56, GinH61 and LysH64 (Table IV). In CDR-H3,TyrH97Ala reduced binding by 11-fold and TyrH100cAla by 8-fold. As inCDR-L3, simultaneous alteration of several CDR-H3 residues to alanineeffected a non-additive, large reduction in binding (cf. variant H3versus TyrH97Ala and TyrH99Ala, Table IV). In addition, the FR-3 residueincluded in the humanization, LysH73, also showed a 5-fold reduction inbinding when changed to alanine or arginine (Table IV).

(c) Re-engineering HulgG1 to Bind to Rhesus CD11a

Both murine MHM24 and HulgG1 showed approximately 1000-fold reduction inbinding to rhesus CD11a: HulgG1 had an EC50 against rhesus CD11a of45.6±40.4 nM (N=16) compared to an EC50 of 0.042±0.072 nM against humanCD11a. Since a primate model is important in evaluating the biology,toxicity, and efficacy of MHM24, improving the binding of HulgG1 torhesus CD11a was deemed advantageous. Initially, the MAb hypervariableregion residues which were important in binding to human CD11a andrhesus CD11a were determined so that those important for the rhesus butnot the human could be altered. Accordingly, the alanine-scan variantswere also assayed against rhesus CD11a on peripheral blood lymphocytes.The most important finding was that one of the multiple-alanine mutationvariants, variant H2, bound 18-fold better to rhesus CD11a than HulgG1(Table IV). However, individual mutations at the three residues includedin variant H2 showed minimal improvement in binding: HisH52Ala, 0.7-foldbetter, SerH53Ala, 0.7-fold better, and SerH55Ala, 1.3-fold worse (TableIV). A series of double mutations at these three residues showed thatthe combination HisH52Ala-SerH53Ala was the best, providing a 77-foldimprovement in binding compared to HulgG1 (cf. variants H2A1, H2A2 andH2A3, Table IV). In addition, the AspH54Ala and GluH56Ala variants alsoeffected a 3-fold improvement over HulgG1 (Table IV), even though AspH54is the most important binding residue in HulgG1 with respect to humanCD11a.

In an attempt to find a single substitution at position H54 which wouldimprove binding to rhesus CD11a but not reduce binding to human CD11a,position H54 was substituted with a variety of amino acids. Allsubstitutions reduced binding by greater than an order of magnitudewhereas the substitution AspH54Asn improved rhesus binding by 10-fold(Table V).

                  TABLE V                                                         ______________________________________                                        Amino acid substitution at AspH54                                                  AspH54  Variant EC50/HUIgG1 EC50.sup.c                                   change   Human CD11a.sup.b    Rhesus                                          to       mean          S. D.  CD11a.sup.c                                     ______________________________________                                        Ala      147           18     0.3                                               Asn 26 1 0.1                                                                  Gln 20 1 4.4                                                                  Glu 16 2 >25                                                                  Ser >100  0.9                                                                 Arg >250  >25                                                                 Lys >100  3.8                                                                 His >300  >25                                                                 Thr >450  >25                                                                 Met >150  >25                                                                 Leu >300  >25                                                               ______________________________________                                         .sup.a EC50 HuIgG1 for human CD11a = 0.042 nM (S. D. = 0.072; N = 15);        EC50 HuIg1 for rhesus CD11a = 45.6 nM (S. D. = 40.4; N = 16).                 .sup.b Values are the mean of two assays.                                     .sup.c Values are for a single assay.                                    

Since non-additive effects were noted for changes at positions H52-H53,these were combined with a variety of changes at positions H54 and H56(Table VI). For all variants, H52 and H53 were alanine. In one series,position H54 was Asn and position H56 was Glu (original), Ala, Asn orGln. None of these variants improved rhesus CD11a binding over the H2A1variant (Table VI). In another series, position H54 was Ala and positionH56 was Glu (original), Ala, Ser or Asn and again all were worse thanvariant H2A1. In the third series, position H54 was Ser and position H56was Glu (original), Ala, Ser or Asn. Two of these variants exhibitedimproved binding to rhesus CD11a compared to the H2A1 variant (H2C11 andH2C12, Table VI). The rhesus CD11a EC50 for these two variants was0.11±0.11 nM (N=9) for H2C11 and 0.19±0.08 nM (N=7) for H2C12. Thesevalues are 3- to 5-fold weaker than the EC50 of HulgG1 for human CD11a(0.042 nM) but are a 240- to 415-fold improvement over the EC50 ofHulgG1 for rhesus CD1a (45.6 nM). H2C12 will hereafter be referred to asRhigG1. Apparent K_(d) values from saturation binding experiments showedthat RhlgG1 bound to rhesus CD11a as well as murine MHM23 bound torhesus CD18 (Table III).

                                      TABLE VI                                    __________________________________________________________________________    Binding of CDR-H2 variants to human and rhesus CD11a                                            Var.EC50/HulgG1 EC50.sup.a                                  Variant           Human                                                                             Rhesus CD11a                                            IgG1 Sequence     CD11a                                                                             Mean  S.D.                                              __________________________________________________________________________    H2C2       A      2.6 >100                                                       - H2C3          A A A N                     >100       >100                   - CDR-H2     M I H P S D S E T R Y         1.0        1.00                    - H2A1          A   A                        10.8        0.013  0.012                                  (N = 10)                                             - H2C1          A   A N                    >100          0.56                                          0.01                                                 - H2C4          A   A N   A               >100          0.38                                           0.06                                                 - H2C5          A   A N   N                 46          0.11                                           0.02                                                 - H2C6          A   A N   Q               >100          0.21                                           0.01                                                 - H2C8          A   A A                      12.7        0.38                 - H2C7          A   A A   A                  2.4        1.03                                           0.05                                                 - H2C10         A   A A   S                 14.2        0.22                                           0.03                                                 - H2C9          A   A A   N                 34.3        0.22                                           0.04                                                 - H2C13         A   A S                                   0.10                                         0.06                                                 - H2C14         A   A S   A                              0.021                                         0.013                                                - H2C12         A   A S   S                              0.004  0.001                                  (N = 7)                                              - H2C11         A   A S   N                 24.9        0.002  0.001                                   (N = 9)                                           __________________________________________________________________________     .sup.a EC50 HulgG1 for human CD11a = 0.042 nM (S.D. = 0.072; N = 15); EC5     HulgG1 for rhesus CD11a = 45.6 nM (S.D. = 40.4; N = 16); all values for       rhesus CD11a are the mean of two independent binding assays except as         noted.                                                                   

For the HulgG1-human CD11a interaction, AspH54 was the most importantresidue (Table IV); changing this residue to other amino acidssignificantly reduced binding with the least reduction occurring forchanges to Glu, Asn and GIn. However, for the HulgG1-rhesus CD11ainteraction, AspH54 was deleterious since changing this residue to Alaor Asn improved binding (Table V). In order to understand thisdifference between binding to human and rhesus CD11a, the latter wascloned from a rhesus PBL library. FIG. 2 shows that rhesus CD11aI-domain differs from human CD11a I-domain at only four positions: 133,189, 197, 308. Previously the human CD11a epitope of MHM24 was mapped toresidues 197-203 (Champe et al., J. Biol. Chem. 270: 1388-1394 (1995))which includes the human Lys197 to rhesus Glu197 change in rhesus.

(d) Keratinocyte Cell Adhesion Assays

Murine MHM24, chimeric IgG1 and HulgG1 were compared in their ability toprevent adhesion of Jurkat cells (human T-cells expressing LFA-1) tonormal human epidermal keratinocytes expressing ICAM-1. All threeantibodies performed similarly (FIG. 3) with similar IC50 values (TableVII).

                                      TABLE VII                                   __________________________________________________________________________    Blocking of cell adhesion by MHM24 variants                                         IC50 Value (nM)                                                                              RhLY:                                                      RhLy.sup.b :HUK HUICAM.sup.c Rh/HuCD11a.sup.d :HUK                          mAb   Jurkat:HuK.sup.a                                                                    Mean                                                                             S. D.                                                                            N  Mean                                                                             S. D.                                                                            N Mean                                                                              S. D.                                                                            N                                         __________________________________________________________________________    murMHM24                                                                            0.09                                                                      HulgG1 0.13                                                                   chlgG1 0.10                                                                   RhlgG1  119 86 4 5.3 4.5 3 4.9 0.2 2                                          MHM23  1.6 1.5 3 1.2   1.4 0.1 2                                            __________________________________________________________________________     .sup.a HuK = normal human epidermal keratinocyte.                             .sup.b RhLy = rhesus lymphocyte.                                              .sup.c HuICAM = recombinant human ICAM1.                                      .sup.d Rh/HuCD11a = human CD11a with rhesus Idomain transfected into huma     293 cells.                                                               

Neither murine nor humanized MHM24 blocked rhesus or cynomolguslymphocytes from adhering to human keratinocytes. When RhIgGl wascompared to the murine anti-human CD18 antibody MHM23 (Hildreth et al.,Eur. J. Immunol. 13: 202-208 (1983); Hildreth et al., J. Immunol. 134:3272-3280 (1985)) in blocking adherence of rhesus lymphocytes to humankeratinocytes, RhlgG1 was 74-fold less efficacious than MHM23 (FIG. 4A,Table VII). However, when recombinant human ICAM-1 was coated on plates(instead of human keratinocytes) RhlgG1 was only 4-fold less efficaciousthan MHM23 (FIG. 4B, Table VII). A chimeric CD11a comprised of humanCD11a in which the I-domain was mutated to rhesus (Val133lle, Arg189Gln,Lys197Glu, Val308Ala) was transfected into human embryonic kidney 293cells. Again, RhlgG1 was only 4-fold down from MHM23 in blocking theseRh/HuCD11a-293 cells from adhering to human keratinocytes (FIG. 4C,Table VII).

Control isotype antibodies for RhlgG1 (humanized anti-p185HER2 antibody;Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992)) and MHM23(murine MAb 354, a murine IgG1 anti-hamster tPA) did not block bindingof rhesus lymphocytes to human keratinocytes or recombinant ICAM-1(FIGS. 4A, 4B) or Rh/HuCD11a to human keratinocytes (FIG. 4C). Thisimplies that the reduced performance of RhlgG1 compared to murine MHM23in the rhesus lymphocyte:human keratinocyte assay was not due to anyunexpected interaction of the human Fc of HulgG1 (compared to the murineFc of MHM23) with the rhesus lymphocytes, which might reduce theconcentration of RhlgG1 available for binding to rhesus CD11a. Therecombinant human ICAM-1 data show that RhlgG1 is binding to the rhesuslymphocytes and preventing adherence almost as well as murine MHM23(FIG. 4B, Table VII). The Rh/HuCD11a-293 data (FIG. 4C, Table VII) showthat RhlgG1 is not binding to targets on the human keratinocytes(compared to HulgG1), which might reduce the concentration of RhlgG1available for binding to rhesus CD11a. In addition, the K_(d) (app) ofRhlgG1 to rhesus leukocytes was similar with (rhesus donor 3) andwithout (rhesus donor 1) addition of 1 mg/ml human IgG1 (Table III);This shows that binding of RhlgG1 is specific to rhesus CD11a.

(e) Mixed Lymphocyte Response Assays

In the MLR, HulgG1 exhibited an IC50 value 2-fold weaker than the murineMHM24 (Table VIII, FIG. 5).

                  TABLE VIII                                                      ______________________________________                                        Mixed lymphocyte response assay results                                                  IC50 Value (nM)                                                    mAb.sup.a  Mean          S. D.  N                                             ______________________________________                                        murMHM24   0.19          0.06   3                                               HuIgG1 0.38 0.14 4                                                            mAb 25.3 3.8 1.0 2                                                            RhIgG1 23.4 11.4 2                                                            MHM23 30.4 24.0 3                                                           ______________________________________                                         .sup.a murMHM24 HuIgG1 and mAb 25.3 tested in human MLR; RhlgG1 and MHM23     tested in rhesus MLR.                                                    

Both the murine and humanized MAbs fared 10- to 20-fold better than MAb25.3, which has been previously tested in vivo (Fischer et al, Blood 77:249-256 (1991); Stoppa et al., Transplant Intl. 4: 3-7 (1991); Hourmantet al., Transplantation 58: 377-380 (1994)). The rhesus-binding variantRhlgG1 exhibited an IC50 value slightly better than murine MHM23 (TableVII). Different responder:stimulator blood donors were used inindependent assays and the results did not vary significantly. The K_(d)of RhlgG1 for rhesus CD11a is about 26-fold down from the K_(d) ofHulgG1 for human CD11a (Table II) and this is reflected in the IC50values derived from the MLR assays (Table VII).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 24                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Le - #u Ala Ala Ser Pro           1               5 - #                 10 - #                 15              - - Gly Glu Thr Ile Ser Ile Asn Cys Arg Ala Se - #r Lys Thr Ile Ser                           20 - #                 25 - #                 30              - - Lys Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gl - #y Lys Thr Asn Lys                           35 - #                 40 - #                 45              - - Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Se - #r Gly Ile Pro Ser                           50 - #                 55 - #                 60              - - Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Ph - #e Thr Leu Thr Ile                           65 - #                 70 - #                 75              - - Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Ty - #r Tyr Cys Gln Gln                           80 - #                 85 - #                 90              - - His Asn Glu Tyr Pro Leu Thr Phe Gly Thr Gl - #y Thr Lys Leu Glu                           95 - #                100 - #                105              - - Leu Lys Arg                                                                      108                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Le - #u Ser Ala Ser Val            1               5 - #                 10 - #                 15              - - Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Se - #r Lys Thr Ile Ser                           20 - #                 25 - #                 30              - - Lys Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gl - #y Lys Ala Pro Lys                           35 - #                 40 - #                 45              - - Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Se - #r Gly Val Pro Ser                           50 - #                 55 - #                 60              - - Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Ph - #e Thr Leu Thr Ile                           65 - #                 70 - #                 75              - - Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Ty - #r Tyr Cys Gln Gln                           80 - #                 85 - #                 90              - - His Asn Glu Tyr Pro Leu Thr Phe Gly Gln Gl - #y Thr Lys Val Glu                           95 - #                100 - #                105              - - Ile Lys Arg                                                                      108                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Le - #u Ser Ala Ser Val            1               5 - #                 10 - #                 15              - - Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Se - #r Gln Ser Ile Ser                           20 - #                 25 - #                 30              - - Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gl - #y Lys Ala Pro Lys                           35 - #                 40 - #                 45              - - Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Se - #r Gly Val Pro Ser                           50 - #                 55 - #                 60              - - Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Ph - #e Thr Leu Thr Ile                           65 - #                 70 - #                 75              - - Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Ty - #r Tyr Cys Gln Gln                           80 - #                 85 - #                 90              - - Tyr Asn Ser Leu Pro Trp Thr Phe Gly Gln Gl - #y Thr Lys Val Glu                           95 - #                100 - #                105              - - Ile Lys Arg                                                                      108                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 121 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Glu Val Gln Leu Gln Gln Pro Gly Ala Glu Le - #u Met Arg Pro Gly            1               5 - #                 10 - #                 15              - - Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gl - #y Tyr Ser Phe Thr                           20 - #                 25 - #                 30              - - Gly His Trp Met Asn Trp Val Arg Gln Arg Pr - #o Gly Gln Gly Leu                           35 - #                 40 - #                 45              - - Glu Trp Ile Gly Met Ile His Pro Ser Asp Se - #r Glu Thr Arg Leu                           50 - #                 55 - #                 60              - - Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Th - #r Val Asp Lys Ser                           65 - #                 70 - #                 75              - - Ser Ser Ser Ala Tyr Met Gln Leu Ser Ser Pr - #o Thr Ser Glu Asp                           80 - #                 85 - #                 90              - - Ser Ala Val Tyr Tyr Cys Ala Arg Gly Ile Ty - #r Phe Tyr Gly Thr                           95 - #                100 - #                105              - - Thr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Th - #r Leu Thr Val Ser                          110  - #               115  - #               120              - - Ser                                                                      121                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 121 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Glu Val Gln Leu Val Glu Ser Gly Gly Gly Le - #u Val Gln Pro Gly            1               5 - #                 10 - #                 15              - - Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gl - #y Tyr Ser Phe Thr                           20 - #                 25 - #                 30              - - Gly His Trp Met Asn Trp Val Arg Gln Ala Pr - #o Gly Lys Gly Leu                           35 - #                 40 - #                 45              - - Glu Trp Val Gly Met Ile His Pro Ser Asp Se - #r Glu Thr Arg Tyr                           50 - #                 55 - #                 60              - - Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Se - #r Val Asp Lys Ser                           65 - #                 70 - #                 75              - - Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Le - #u Arg Ala Glu Asp                           80 - #                 85 - #                 90              - - Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ile Ty - #r Phe Tyr Gly Thr                           95 - #                100 - #                105              - - Thr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Le - #u Val Thr Val Ser                          110  - #               115  - #               120              - - Ser                                                                      121                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 113 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Glu Val Gln Leu Val Glu Ser Gly Gly Gly Le - #u Val Gln Pro Gly            1               5 - #                 10 - #                 15              - - Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gl - #y Phe Thr Phe Ser                           20 - #                 25 - #                 30              - - Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pr - #o Gly Lys Gly Leu                           35 - #                 40 - #                 45              - - Glu Trp Val Ser Val Ile Ser Gly Asp Gly Gl - #y Ser Thr Tyr Tyr                           50 - #                 55 - #                 60              - - Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Se - #r Arg Asp Asn Ser                           65 - #                 70 - #                 75              - - Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Le - #u Arg Ala Glu Asp                           80 - #                 85 - #                 90              - - Thr Ala Val Tyr Tyr Cys Ala Arg Gly Phe As - #p Tyr Trp Gly Gln                           95 - #                100 - #                105              - - Gly Thr Leu Val Thr Val Ser Ser                                                          110  - #       113                                             - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 184 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Lys Gly Asn Val Asp Leu Val Phe Leu Phe As - #p Gly Ser Met Ser            1               5 - #                 10 - #                 15              - - Leu Gln Pro Asp Glu Phe Gln Lys Ile Leu As - #p Phe Met Lys Asp                           20 - #                 25 - #                 30              - - Val Met Lys Lys Leu Ser Asn Thr Ser Tyr Gl - #n Phe Ala Ala Val                           35 - #                 40 - #                 45              - - Gln Phe Ser Thr Ser Tyr Lys Thr Glu Phe As - #p Phe Ser Asp Tyr                           50 - #                 55 - #                 60              - - Val Lys Arg Lys Asp Pro Asp Ala Leu Leu Ly - #s His Val Lys His                           65 - #                 70 - #                 75              - - Met Leu Leu Leu Thr Asn Thr Phe Gly Ala Il - #e Asn Tyr Val Ala                           80 - #                 85 - #                 90              - - Thr Glu Val Phe Arg Glu Glu Leu Gly Ala Ar - #g Pro Asp Ala Thr                           95 - #                100 - #                105              - - Lys Val Leu Ile Ile Ile Thr Asp Gly Glu Al - #a Thr Asp Ser Gly                          110  - #               115  - #               120              - - Asn Ile Asp Ala Ala Lys Asp Ile Ile Arg Ty - #r Ile Ile Gly Ile                          125  - #               130  - #               135              - - Gly Lys His Phe Gln Thr Lys Glu Ser Gln Gl - #u Thr Leu His Lys                          140  - #               145  - #               150              - - Phe Ala Ser Lys Pro Ala Ser Glu Phe Val Ly - #s Ile Leu Asp Thr                          155  - #               160  - #               165              - - Phe Glu Lys Leu Lys Asp Leu Phe Thr Glu Le - #u Gln Lys Lys Ile                          170  - #               175  - #               180              - - Tyr Val Ile Glu                                                                      184                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 184 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Lys Gly Asn Val Asp Leu Ile Phe Leu Phe As - #p Gly Ser Met Ser            1               5 - #                 10 - #                 15              - - Leu Gln Pro Asp Glu Phe Gln Lys Ile Leu As - #p Phe Met Lys Asp                           20 - #                 25 - #                 30              - - Val Met Lys Lys Leu Ser Asn Thr Ser Tyr Gl - #n Phe Ala Ala Val                           35 - #                 40 - #                 45              - - Gln Phe Ser Thr Ser Tyr Lys Thr Glu Phe As - #p Phe Ser Asp Tyr                           50 - #                 55 - #                 60              - - Val Lys Gln Lys Asp Pro Asp Ala Leu Leu Gl - #u His Val Lys His                           65 - #                 70 - #                 75              - - Met Leu Leu Leu Thr Asn Thr Phe Gly Ala Il - #e Asn Tyr Val Ala                           80 - #                 85 - #                 90              - - Thr Glu Val Phe Arg Glu Glu Leu Gly Ala Ar - #g Pro Asp Ala Thr                           95 - #                100 - #                105              - - Lys Val Leu Ile Ile Ile Thr Asp Gly Glu Al - #a Thr Asp Ser Gly                          110  - #               115  - #               120              - - Asn Ile Asp Ala Ala Lys Asp Ile Ile Arg Ty - #r Ile Ile Gly Ile                          125  - #               130  - #               135              - - Gly Lys His Phe Gln Thr Lys Glu Ser Gln Gl - #u Thr Leu His Lys                          140  - #               145  - #               150              - - Phe Ala Ser Lys Pro Ala Ser Glu Phe Val Ly - #s Ile Leu Asp Thr                          155  - #               160  - #               165              - - Phe Glu Lys Leu Lys Asp Leu Phe Thr Glu Le - #u Gln Lys Lys Ile                          170  - #               175  - #               180              - - Tyr Ala Ile Glu                                                                      184                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Lys His Val Lys His Met Leu                                                1               5 - #      7                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Gly Tyr Ser Phe Thr Gly His Trp Met Asn                                    1               5 - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Met Ile His Pro Ser Asp Ser Glu Thr Arg Ty - #r Asn Gln Lys Phe            1               5 - #                 10 - #                 15              - - Lys Asp                                                                       17                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Gly Ile Tyr Phe Tyr Gly Thr Thr Tyr Phe As - #p Tyr                        1               5 - #                 10 - #     12                          - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Arg Ala Ser Lys Thr Ile Ser Lys Tyr Leu Al - #a                            1               5 - #                 10 - # 11                              - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Ser Gly Ser Thr Leu Gln Ser                                                1               5 - #      7                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Gln Gln His Asn Glu Tyr Pro Leu Thr                                        1               5 - #              9                                         - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Pro Lys Asn Ser Ser Met Ile Ser Asn Thr Pr - #o                            1               5 - #                 10 - # 11                              - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - His Gln Ser Leu Gly Thr Gln                                                1               5 - #      7                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - His Gln Asn Leu Ser Asp Gly Lys                                            1               5 - #          8                                             - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - His Gln Asn Ile Ser Asp Gly Lys                                            1               5 - #          8                                             - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Val Ile Ser Ser His Leu Gly Gln                                            1               5 - #          8                                             - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - CACTTTGGAT ACCGCGTCCT GCAGGT          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - CATCCTGCAG GTCTGCCTTC AGGTCA          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Met Ile Ala Pro Ala Ser Ser Ser Thr Arg Ty - #r Asn Gln Lys Phe            1               5 - #                 10 - #                 15              - -  Lys Asp                                                                       17                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 121 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Glu Val Gln Leu Val Glu Ser Gly Gly Gly Le - #u Val Gln Pro Gly            1               5 - #                 10 - #                 15              - - Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gl - #y Tyr Ser Phe Thr                           20 - #                 25 - #                 30              - - Gly His Trp Met Asn Trp Val Arg Gln Ala Pr - #o Gly Lys Gly Leu                           35 - #                 40 - #                 45              - - Glu Trp Val Gly Met Ile Ala Pro Ala Ser Se - #r Ser Thr Arg Tyr                           50 - #                 55 - #                 60              - - Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Se - #r Val Asp Lys Ser                           65 - #                 70 - #                 75              - - Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Le - #u Arg Ala Glu Asp                           80 - #                 85 - #                 90              - - Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ile Ty - #r Phe Tyr Gly Thr                           95 - #                100 - #                105              - - Thr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Le - #u Val Thr Val Ser                          110  - #               115  - #               120              - - Ser                                                                      121                                                                          __________________________________________________________________________

What is claimed is:
 1. A humanized anti-CD11a antibody which bindsspecifically to human CD11a I-domain, said antibody containing a heavychain variable region comprising the amino acid sequence of (a) CDR2(SEQ ID NO:11), (b) CDR1 (SEQ ID NO:10), CDR2 (SEQ ID NO:11) and CDR3(SEQ ID NO:12) or (c) SEQ ID NO:5.
 2. The humanized anti-CD11a antibodyof claim 1 having a heavy chain variable region comprising the aminoacid sequence of CDR1 (SEQ ID NO:10), CDR2 (SEQ ID NO:11) and CDR3 (SEQID NO:12) of humanized antibody MHM24 (F(ab)-8.
 3. The humanizedanti-CD11a antibody of claim 2 comprising the amino acid sequence of SEQID NO:5.
 4. The humanized anti-CD11a antibody of claim 1 having a lightchain variable region comprising the amino acid sequence of CDR1 (SEQ IDNO:13), CDR2 (SEQ ID NO:14) and CDR3 (SEQ ID NO:15) of humanized MHM24F(ab)-8.
 5. The humanized anti-CD11a antibody of claim 4 comprising theamino acid sequence of SEQ ID NO:2.
 6. The humanized anti-CD11a antibodyof claim 1 having a light chain variable region comprising the aminoacid sequence of SEQ ID NO:5 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:2.
 7. The humanizedanti-CD11a antibody of claim 1 which is a full length antibody.
 8. Thehumanized anti-CD11a antibody of claim 7 which is a human IgG.
 9. Thehumanized anti-CD11a antibody of claim 1 which is an antibody fragment.10. The humanized anti-CD11a antibody of claim 9 wherein the antibodyfragment is a F(ab')₂.
 11. A labeled antibody comprising the humanizedanti-CD11a antibody of claim 1 bound to a detectable label.
 12. Animmobilized antibody comprising the humanized anti-CD11a antibody ofclaim 1 bound to a solid phase.
 13. A conjugate comprising the humanizedanti-CD11a antibody of claim 1 bound to a cytotoxic agent.
 14. A methodfor determining the presence of CD11a protein comprising exposing asample suspected of containing the CD11a protein to the humanizedanti-CD11a antibody of claim 1 and determining binding of said antibodyto the sample.
 15. A kit comprising the humanized anti-CD11a antibody ofclaim 1 and instructions for using the humanized anti-CD11a antibody todetect the CD11a protein.
 16. Isolated nucleic acid encoding thehumanized anti-CD11a antibody of claim
 1. 17. A vector comprising thenucleic acid of claim
 16. 18. A host cell comprising the vector of claim17.
 19. A process of producing a humanized anti-CD11a antibodycomprising culturing the host cell of claim 18 so that the nucleic acidis expressed.
 20. The process of claim 19 further comprising recoveringthe humanized anti-CD11a antibody from the host cell culture.
 21. Theprocess of claim 20 wherein the humanized anti-CD11a antibody isrecovered from the host cell culture medium.
 22. The humanizedanti-CD11a antibody of claim 1, having at least one of the followingproperties:(a) binds human CD11a with a K_(d) value of no more thanabout 1×10⁻⁸ M, (b) has an IC50 (nM) value of no more than about 1 nMfor preventing adhesion of Jurkat cells to normal human epidermalkeratinocytes expressing ICAM-1, or (c) has an IC50 (nM) value of nomore than about 1 nM in a mixed lymphocyte response assay.
 23. Thehumanized anti-CD11a antibody of claim 1, having human kappa I consensuslight chain framework residue 66L.
 24. The humanized anti-CD11a antibodyof claim 1, having all human kappa I consensus light chain frameworkresidues.
 25. The humanized anti-CD11a antibody of claim 1, having humanV_(H) subgroup III consensus heavy chain framework residue 93H.
 26. Ahumanized anti-CD11a antibody which binds specifically to human CD11aI-domain containing a variable domain having a non-human CDRincorporated into a V_(H) subgroup III consensus human antibody variabledomain, and further comprising an amino acid substitution at a siteselected from the group consisting of 27H, 28H, 30H, 49H, 71H and 73H.27. The humanized anti-CD11a antibody of claim 26, having at least oneof the following properties:(a) binds human CD11a with a K_(d) value ofno more than about 1×10⁻⁸ M, (b) has an IC50 (nM) value of no more thanabout 1 nM for preventing adhesion of Jurkat cells to normal humanepidermal keratinocytes expressing ICAM-1, or (c) has an IC50 (nM) valueof no more than about 1 nM in a mixed lymphocyte response assay.
 28. Thehumanized anti-CD11a antibody of claim 26, having human kappa Iconsensus light chain framework residue 66L.
 29. The humanizedanti-CD11a antibody of claim 26, having all human kappa I consensuslight chain framework residues.
 30. The humanized anti-CD11a antibody ofclaim 26, having human V_(H) subgroup III consensus heavy chainframework residue 93H.